Variant B7 co-stimulatory molecules

ABSTRACT

The invention provides polynucleotides and polypeptides encoded therefrom having advantageous properties, including an ability of the polypeptides to preferentially bind a CD28 or CTLA-4 receptor at a level greater or less than the ability of human B7-1 to bind CD28 or CTLA-4, or to induce or inhibit altered level of T cell proliferation response greater compared to that generated by human B7-1. The polypeptides and polynucleotides of the invention are useful in therapeutic and prophylactic treatment methods, gene therapy applications, and vaccines.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This work was supported in part by a grant from the Defense AdvancedResearch Projects Agency (DARPA) (Grant No. N65236-98-1-5401). TheGovernment may have certain rights in this invention.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates generally to polynucleotides andpolypeptides encoded therefrom, as wells as vectors, cells, antibodies,and methods for using and producing the polynucleotides andpolypeptides.

BACKGROUND OF THE INVENTION

T cells are a crucial component of the immune system. Not only is T cellactivation required for all specific immune responses against infectiousagents, but T cells also play an important role in tumor immunity and inautoimmune and allergic diseases. T cell activation is initiated when Tcells recognize their specific antigen (Ag) in the context of majorhistocompatibility complex (MHC) molecules. T cell activation is wellknown by those of ordinary skill in the art and is characterized by suchthings as, e.g., cytokine synthesis, induction of various activationmarkers such as CD25 (interleukin-2 (IL-2) receptor), etc. CD4+ T cellsrecognize their immunogenic peptides in the context of MHC class IImolecules, whereas CD8+ T cells recognize their immunogenic peptides inthe context of MHC class I molecules. For induction of T cellactivation, cytokine synthesis or effector function, a second signal,mediated through CD28, is required. Two ligands for CD28 are B7-1 (CD80)and B7-2 (CD86). B7-1 and B7-2 are termed co-stimulatory molecules andare typically expressed on professional antigen-presenting cells (APCs).In addition to binding the CD28 receptor, B7-1 and B7-2 also bind theCTLA-4 (CD152) receptor on T cells.

B7 molecules mediate both positive and negative signals to T cells bybinding to CD28 and CTLA-4 (CD152) molecules on T cells. CTLA-4 is anegative regulator of the immune system. In general, wild-type (WT)B7-1, e.g., human B7-1, preferentially binds CTLA-4 more strongly thanit binds CD28. Typically, wild-type B7-1, e.g., human B7-1, binds CTLA-4with about 100 times greater affinity than it binds CD28. Binding ofB7-1 or B7-2 to CTLA-4 suppresses activation of T cells, resulting inreduced T cell proliferation and cytokine production (see, e.g.,Walunas, T. L. et al. (1994) Immunity 1(5):405–413; Alegre, M. L. et al.(1998) J Immunol 161(7):3347–3356). Interaction between B7-1 or B7-2 andCTLA-4 expressed on T cells down-regulates T cell responses and raisesthresholds required for activation by CD28. Blockade of CTLA-4/ligandinteractions can also augment in vivo tumor immunity (Leach, D. et al.(1996) Science 271:1734–1736). Consequently, CD28 and CTLA-4 play apivotal role in the regulation of T cell activation and both areessential for proper functioning of the immune system. For example, CD28deficient mice are severely immunodeficient and show poor antigenspecific T cell responses, while CTLA-4 deficient mice die oflymphoproliferative disease, show T cell expansion mediated by CD28signaling and have a lack of down-regulation of T cell receptorsignaling. Upon ligation by the co-stimulatory molecules B7-1 or B7-2,CD28 mediates a co-stimulatory signal that synergizes with T cellreceptor signaling to induce, e.g., proliferation, cytokine productionand effector functions by both CD4+ and CD8+ T cells(proliferation/activation). Ligation of CTLA-4 with B7-1 (CD80) or B7-2(CD86), however, dampens the CD80 or CD86 activating signal throughCD28, resulting in down-regulation of T cell activation. CD28 ligationreduces the inhibition mediated through the CTLA-4 signaling. CTLA-4ligation mediates tolerance and anergy.

CD28 and CTLA-4 are both involved in the generation of an immuneresponse to genetic vaccinations (e.g., nucleic acid vaccinations (NAV),DNA vaccinations, and viral vectors). CD28 deficient mice are unable tomount T cell or antibody responses against Beta-galactosidase (Beta-gal)when immunized with a plasmid encoding the Beta-gal gene, and CTLA-4ligation suppresses the antibody response to Beta-gal in immunizedwild-type mice (Horspool, J. et al. (1998), J Immunol 160:2706–2714).Expression of B7-1 on human myeloma cells (Wendtner, C. et al. (1997)Gene Therapy 4(7):726–735), murine mammary tumors (Martin-Fontecha, A.et al.(2000) J Immunol 164(2):698–704) or murine sarcoma (Indrova et al.(1998) Intl J Onc 12(2):387–390) enhances anti-tumor immunity.Furthermore, transfection of human APCs with retroviral vectors encodingB7-1 and tumor antigens induces a stronger cytotoxic T-lymphocyte (CTL)response than transfection with similar vectors encoding the tumorantigens alone (Zajac, P. et al. (1998) Cancer Res 58(20):4567–4571).Anti-viral responses are also modulated by co-stimulatory molecules. Forexample, DNA vaccination of chimpanzees and mice with HIV antigens inconjunction with B7-2 augmented anti-viral responses (Kim, J. et al.(1998) Vaccine 16(19):1828–1835; Tsuji et al. (1997) Eur J Immunol27(3):782–787).

The binding properties of B7-1 and B7-2 have limited their usefulness inclinical applications. The present invention addresses needs formolecules having varied abilities to preferentially bind to and/orsignal through either CD28 or CTLA-4 receptor and methods of using suchmolecules for selected and differential manipulation of T cell responsesin vitro, ex vivo, and in vivo methods. Such molecules would be ofbeneficial use in a variety of applications, including, e.g.,therapeutic and prophylactic treatments and vaccinations. The presentinvention fulfills these and other needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel co-stimulatorymolecules (abbreviated as “NCSM”) molecules, including polypeptides andproteins, related fusion polypeptide or fusion protein molecules, orfunctional equivalents thereof, homologues, and fragments of saidpolypeptide and protein molecules or equivalents, analogs, orderivatives thereof, and vectors, cells, and compositions comprisingsuch NCSM molecules. The invention also provides nucleic acids encodingany of these polypeptides, proteins, fragments or variants thereof. Inaddition, the invention provides vectors, cells, and compositionscomprising such nucleic acids, and uses of such NCSM polypeptides andNCSM nucleic acids; and other features are apparent upon further review.

Generally speaking, a “co-stimulatory molecule” refers to a moleculethat acts in association or conjunction with, or is involved with, asecond molecule or with respect to an immune response in aco-stimulatory pathway. In one aspect, a co-stimulatory molecule may bean immunomodulatory molecule that acts in association or conjunctionwith, or is involved with, another molecule to stimulate or enhance animmune response. In another aspect, a co-stimulatory molecule isimmunomodulatory molecule that acts in association or conjunction with,or is involved with, another molecule to inhibit or suppress an immuneresponse. A “co-stimulatory molecule” need not act simultaneously withor by the same mechanism as the second molecule. The term “NCSM” inreference to a molecule is not intended to limit the molecule to onlythose molecules that have positive co-stimulatory properties (e.g., thatstimulate or augment T cell proliferation). In the initial recombinationprocedures described below, libraries of recombinant molecules weregenerated by recombining nucleotide sequences of parental co-stimulatorymolecules (CSM) as discussed herein. As shown by the data and analysespresented herein, novel recombinant molecules having a variety ofproperties were identified and selected. For example, polypeptide andnucleic acid molecules that enhance an immune response, such as byinducing T cell activation or proliferation (e.g., agonists), andmolecules that down-regulate or inhibit an immune response, such as byinhibiting T cell activation or proliferation (e.g., antagonists) wereidentified and selected. Further, molecules that preferentially bindand/or signal through either or both the CD28 and CTLA-4 receptors wereidentified and selected. Thus, the term “NCSM” refers to aco-stimulatory molecule and is not limited to molecules having theco-stimulatory properties of the parent sequences, but is intended torefer collectively to all polypeptides of the invention, and nucleicacids encoding them, and other embodiments as described herein, unlessspecifically noted otherwise.

The term “NCSM” also includes variants, mutants, derivatives, andfragments of: 1) B7-1 and B7-2 polypeptides and nucleic acids, and 2)B7-1 and B7-2 polypeptides and nucleic acids of the Artiodactyla family(including, e.g., bovine B7-1 and B7-2), including all such polypeptidevariants (and nucleic acids encoding such polypeptide variants) thatexhibit properties similar or equivalent to the properties of theCD28BPs or CTLA-4BPs described herein. For example, the term includesB7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 and B7-2 polypeptidevariants (and nucleic acids encoding such polypeptide variants) of theinvention, wherein such polypeptide variants have a CD28/CTLA-4 bindingaffinity ratio about equal to, equal to, or greater than the CD28/CTLA-4binding affinity ratio of hB7-1 or hB7-2 and/or an ability to induce aT-cell proliferation, and/or a T-cell activation response about equalto, equal to, or greater than that of hB7-1. The term “NCSM” also isintended to include B7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 andB7-2 polypeptide variants (and nucleic acids encoding such polypeptidevariants), wherein such polypeptide variants have a CTLA-4/CD28 bindingaffinity ratio about equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of hB7-1 or hB7-2, and/or an ability to induce a T-cellproliferation and/or T-cell activation response about equal to or lessthan that of hB7-1 or hB7-2.

In one aspect, the invention includes isolated or recombinant NCSMpolypeptides, variants, homologues, derivatives, analogs, and fragmentsthereof. The invention includes recombinant NCSM polypeptides havingvaried abilities to preferentially bind to and/or signal through CD28and/or CTLA-4 receptor and provide for selected and differentialmanipulation of T cell responses in vitro, ex vivo, and in vivo. Theinvention also includes isolated or recombinant NCSM nucleic acids,variants, homologues, derivatives, analogs, and fragments thereof thatencode polypeptides having varied abilities and uses described above.Such NCSM polypeptide and polynucleotides are useful in a variety ofapplications, including e.g., therapeutic and prophylactic treatmentmethods, vaccinations, and diagnostic assays described below. Theinvention also provides NCSM polypeptides, and polynucleotide encodingsuch polypeptides, that strongly or preferentially bind at least one ofCD28 or CTLA-4, but do not effectuate signaling; such molecules areuseful in methods as potential antagonists of endogenous molecules, suchas e.g., endogenous co-stimulatory molecules. Further, the inventionprovides NCSM polypeptides, and polynucleotides encoding them, havingimproved or altered receptor/ligand binding affinities and methods ofusing such molecules, including in pharmaceutical, prophylactic,therapeutic, vaccine, and diagnostic applications.

In one aspect, the invention provides an isolated or recombinantpolypeptide comprising an amino acid sequence of an extracellular domain(ECD) amino acid sequence having at least about 75% amino acid sequenceidentity to an extracellular domain amino acid sequence of, or thefull-length sequence of, at least one of SEQ ID NOS:48–68, 174–221,283–285, and 290–293, and not being a naturally-occurring extracellulardomain amino acid sequence, and wherein said polypeptide has aCD28/CTLA-4 binding affinity ratio about equal to or greater than theCD28/CTLA-4 binding affinity ratio of human B7-1 and/or has an abilityto induce a T-cell proliferation and/or T-cell activation response aboutequal to or greater than that of hB7-1. Some such polypeptides induceT-cell proliferation or T-cell activation or both T-cell proliferationand T-cell activation. In some embodiments, the T cell activation orproliferation response is at least about equal to or greater than thatcaused by WT hB7-1.

Some such polypeptides of the invention may comprise an amino acidsequence having 75% identity to a full-length polypeptide sequence ofSEQ ID NOS:48–68, 174–221, 283–285, and 290–293. Such polypeptides maybe expressed on the surface of a cell membrane (e.g., followingtransfection of the cell with a nucleic acid that encodes saidpolypeptide) or associated with or bound to a cell membrane, or form anintegral membrane protein (e.g., by further comprising a transmembranedomain amino acid sequence). Through such expression, such polypeptidestypically become integral membrane proteins. Preferably, suchcell-expressed polypeptides, membrane-associated, or membrane-boundpolypeptides, have an ability to induce a T cell proliferation responsethat is equal to or greater than that induced by hB7-1.

Other such polypeptides modulate T-cell activation, but do not induceproliferation of purified T-cells activated by soluble anti-CD3 mAbs.

In one embodiment, the isolated or recombinant polypeptide comprises anECD that has at least about 90% amino acid sequence identity to an ECDor the full-length sequence of at least one of SEQ ID NOS:48–68,174–221, 283–285, and 290–293. In another embodiment, the ECD amino acidsequence comprises an amino acid subsequence of at least one of SEQ IDNOS:48–68, 174–221, 283–285, and 290–293.

Some such isolated or recombinant polypeptides are soluble (e.g., notcell membrane-bound or membrane-associated or forming an integral partof a cell membrane). The invention includes monomeric and multimeric (oraggregated) forms of such soluble polypeptides. A soluble monomercomprises one soluble polypeptide of the invention (e.g., one solubleNCSM-ECD), while a soluble multimer or soluble aggregate comprises atleast two soluble polypeptides of the invention (e.g., two solubleECDs). The multimer or aggregate may be formed by cross-linking or othermeans to link polypeptide sequences. Such polypeptides may comprise afusion protein comprising at least one additional amino acid sequence,such as an Ig polypeptide. Some such soluble polypeptides, in thepresence of a population of activated T cells, have an ability to inducea T cell proliferation response, in the presence of a population ofactivated T cells, that is less than the T cell proliferation responseinduced by a soluble human B7-1 polypeptide in the presence of apopulation of activated T cells.

Some such isolated or recombinant polypeptides further comprise at leastone of a signal peptide domain, transmembrane domain (TMD), and/orcytoplasmic domain(CD), including, e.g., wherein such domain is a WT,variant, or mutant domain of a co-stimulatory polypeptide, including,e.g., a recombinant domain derived from a mammalian B7-1 or B7-2. In oneembodiment, the isolated or recombinant polypeptide comprises an aminoacid subsequence, including any of a signal peptide, TMD, or CD of anyof SEQ ID NOS:48–68, 174–221, 283–285, and 290–293. Nucleic acidsencoding such polypeptides and domains are also provided.

In another aspect, the invention provides an isolated or recombinantpolypeptide, which polypeptide comprises a non-naturally-occurring aminoacid sequence encoded by a nucleic acid comprising a polynucleotidesequence selected from the group of: (a) a polynucleotide sequenceselected from SEQ ID NOS:1–21 and 95–142, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:48–68, 174–221, 283–285, and290–293, or a complementary polynucleotide sequence thereof; (c) apolynucleotide sequence which, but for the degeneracy of the geneticcode, hybridizes under at least stringent or highly stringent conditionsover substantially the entire length of polynucleotide sequence (a) or(b); (d) a polynucleotide sequence comprising all or a nucleotidefragment of (a), (b), or (c), wherein the nucleotide fragment encodes apolypeptide having a CD28/CTLA-4 binding affinity ratio about equal toor greater than the CD28/CTLA-4 binding affinity ratio of human B7-1,and/or has an ability to induce a T-cell proliferation and/or T-cellactivation response about equal to or greater than that of hB7-1; (e) apolynucleotide sequence encoding a polypeptide, the polypeptidecomprising an amino acid sequence which is substantially identical overat least about 150 contiguous amino acid residues of any one of SEQ IDNOS:48–68, 174–221, 283–285, and 290–293; and (f) a polynucleotidesequence encoding a polypeptide that has a CD28/CTLA-4 binding affinityratio about equal to or greater than the CD28/CTLA-4 binding affinityratio of human B7-1, and/or has an ability to induce T-cellproliferation or activation or both that is about equal to or greaterthan that induced by hB7-1, which polynucleotide sequence has at leastabout 70% amino acid sequence identity to at least one polynucleotidesequence of (a), (b), (c), or (d).

Also provided is an isolated or recombinant polypeptide comprising anamino acid sequence according to the formula:MGHTM-X6-W-X8-SLPPK-X14-PCL-X18-X19-X20-QLLVLT-X27-LFYFCSGITPKSVTKRVKETVMLSCDY-X55-TSTE-X60-LTSLRIYW-X69-KDSKMVLAILPGKVQVWPEYKNRTITDMNDN-X101-RIVI-X106-ALR-X110-SD-X113-GTYTCV-X120-QKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVP-X149-X150-X151-DLGNPSPNIRRLICS-X167-X168-X169-GFPRPHL-X177-WLENGEELNATNTT-X192-SQDP-X197-T-X199-LYMISSEL-X208-FNVTNN-X215-SI-X218-CLIKYGEL-X227-VSQIFPWSKPKQEPPIDQLPF-X249-VIIPVSGALVL-X261-A-X263-VLY-X267-X268-ACRH-X273-ARWKRTRRNEETVGTERLSPIYLGSAQSSG (SEQ ID NO:284), or a subsequence thereof comprising anextracellular domain, wherein position X6 is Lys or Glu; position X8 isArg or Gly; position X14 is Arg or Cys; position X18 is Trp or Arg;position X19 is Pro or Leu; position X20 is Ser or Pro; position X27 isAsp or Gly; position X55 is Asn or Ser; position X60 is Glu or Lys;position X69 is Gln or Arg; position X101 is Pro or Leu; position X106is Leu or Gln; position X110 is Pro or Leu; position X113 is Lys or Ser;position X120 is Val or Ile; position X124 is Val or Asp; position X135is Thr or Ala; position X149 is Thr, Ser, or del; position X150 is Ileor del; position X151 is Asn or Thr; position X167 is Thr or del;position X169 is Ser or del; position X169 is Gly or del; position X177is Cys or Tyr; position X192 is Val or Leu; position X197 is Gly or Glu;position X199 is Glu or Lys; position X208 is Gly or Asp; position X215is His or Arg; position X218 is Ala or Val; position X227 is Ser or Leu;position X249 is Trp, Leu, or Arg; position X261 is Ala or Thr; positionX263 is Val, Ala, or Ile; position X267 is Arg or Cys; position X268 isPro or Leu; and position X273 is Gly or Val. Typically, the ECDcomprises at least about amino acids 35 to 244 or amino acids 35 to 255of SEQ ID NO:284. Some such polypeptides have a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than the CD28/CTLA-4 bindingaffinity ratio of human B7-1, and/or has an ability to induce T-cellproliferation or activation or both that is about equal to or greaterthan that induced by hB7-1.

In yet another aspect, the invention provides an isolated or recombinantpolypeptide comprising a subsequence of an amino acid sequence set forthin any of SEQ ID NOS:48–68, 174–182, 184–221, 283–285, and 290–293,wherein the subsequence is the extracellular domain of said amino acidsequence.

A soluble form of an NCSM polypeptide or a human B7-1 polypeptidetypically comprises a polypeptide, comprising at least one ECD, which isnot expressed on the surface of a cell, is not embedded in a cellmembrane, and/or is not associated with a lipid bilayer. Such solublepolypeptides may contain a TD or a fragment thereof such that thepolypeptide remains soluble (e.g., is not embedded membrane as anintegral membrane protein or associated with a lipid bilayer). Suchsoluble polypeptides may also include one or more additional amino acidsequences, such as an Fc portion of an Ig (e.g., IgG). Such solublepolypeptide may comprise an ECD monomer, ECD-Ig fusion protein monomer,ECD multimer or aggregate, or ECD-Ig fusion protein multimer oraggregate (see data and discussion below).

Also provided are isolated or recombinant polypeptides, each of whichcomprise an amino acid sequence of an extracellular domain, wherein saidextracellular domain amino acid sequence has at least about 75% aminoacid sequence identity to an extracellular domain amino acid sequence ofat least one of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, and isnot a naturally-occurring extracellular domain amino acid sequence,wherein such polypeptide, in the presence of activated T cells, has anability to down-regulate or inhibit a T cell proliferation responsecompared to the response caused by soluble hB7-1 (e.g., hB7-1-ECD orhB7-1-ECD-Ig) in the presence of activated T cells. The polypeptide canbe either a soluble ECD monomer or a soluble ECD-Ig fusion protein. Suchpolypeptides may comprise a fusion protein comprising at least oneadditional polypeptide, such as, e.g., an Ig polypeptide.

The invention also includes each nucleic acid comprising apolynucleotide sequence (including degenerate sequences) that encodeseach such isolated or recombinant polypeptide comprising a solublepolypeptide as described above, and a complementary polynucleotidesequence thereof. Polynucleotide sequences that, but for the degeneracyof the genetic code, hybridizes under at least stringent conditions oversubstantially the entire length of each such nucleic acid are alsoincluded.

The invention further provides isolated or recombinant polypeptidescomprising an amino acid sequence having at least about 95% amino acidsequence identity to a full-length sequence of at least one of SEQ IDNOS:69–92, 222–252, 286–289, or to a subsequence thereof comprising theextracellular domain, wherein said sequence (a) is a nonnaturally-occurring sequence, and (b) comprises at least one of: Gly atposition 2; Thr at position 4; Arg at position 5; Gly at position 8; Proat position 12; Met at position 25; Cys at position 27; Pro at position29; Leu at position 31; Arg at position 40; Leu at position 52; His atposition 65; Ser at position 78; Asp at position 80; Tyr at position 87;Lys at position 120; Asp at position 122; Lys at position 129; Met atposition 135; Phe at position 150; Ile at position 160; Ala at position164; His at position 172; Phe at position 174; Leu at position 176; Asnat position 178; Asn at position 186; Glu at position 194; Gly atposition 196; Thr at position 199; Ala at position 210; His at position212; Arg at position 219; Pro at position 234; Asn at position 241; Leuat position 244; Thr at position 250; Ala at position 254; Tyr/ atposition 265; Arg at position 266; Glu at position 273; Lys at position275; Ser at position 276; an amino acid deletion at position 276; or Thrat position 279, wherein the position number corresponds to that of thehuman B7-1 amino acid sequence (SEQ ID NO:278), wherein said polypeptidehas a CTLA-4/CD28 binding affinity ratio about equal to or greater thanthe CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an abilityto induce a T-cell proliferation or T-cell activation response aboutequal to or less than that of hB7-1. A subsequence comprises signalpeptide, EDC, TMD, or CD. Each such polypeptide may further comprise atleast one additional amino acid sequence, including, e.g., a sequencecorresponding to a signal peptide, TMD, ECD, or CD.

In another aspect, the invention provides isolated or recombinantpolypeptides, each comprising an amino acid sequence that differs fromthe amino acid sequence of a primate B7-1, wherein the differencebetween the amino acid sequence of the polypeptide and the amino acidsequence of the primate B7-1 comprises a different amino acid at atleast one amino acid residue position selected from the group consistingof 12, 25, 27, 29, 40, 52, 65, 122, 129, 135, 164, 174, 196, 199, 210,219, 234, 241, 254, 275, 276, and 279, wherein the amino acid residuepositions correspond to the amino acid residue positions in the aminoacid sequence of human B7-1 of SEQ ID NO:278. For some suchpolypeptides, the different amino acid comprises at least one of: Pro atposition 12; Met at position 25; Cys at position 27; Pro at position 29;Arg at position 40; Leu at position 52; His at position 65; Asp atposition 122; Lys at position 129; Met at position 135; Ala at position164; Phe at position 174; Gly at position 196; Thr at position 199; Alaat position 210; Arg at position 219; Pro at position 234; Asn atposition 241; Ala at position 254; Lys at position 275; Ser at position276; or Thr at position 279. Preferably, for some such polypeptides, thedifferent amino acid is His at position 65.

In another aspect, the invention provides isolated or recombinantpolypeptides comprising an amino acid sequence that differs from aprimate B7-1 sequence in at least one mutation selected from: Ser 12Pro; Leu 25 Met; Gly 27 Cys; Ser 29 Pro; Lys 40 Arg; His 52 Leu; Tyr 65His; Glu 122 Asp; Glu 129 Lys; Thr 135 Met; Thr 164 Ala; Ser 174 Phe;Glu 196 Gly; Ala 199 Thr; Thr 210 Ala; Lys 219 Arg; Thr 234 Pro; Asp 241Asn; Val 254 Ala; Arg 275 Lys; Arg 276 Ser; or Arg 279 Thr; the mutationbeing indicated relative to human B7-1 with the amino acid sequenceshown in SEQ ID NO:278, wherein said sequence does not occur in nature,and wherein said polypeptide has a CTLA-4/CD28 binding affinity ratioabout equal to or greater than the CTLA-4/CD28 binding affinity ratio ofhuman B7-1, and/or an ability to induce a T-cell proliferation and/orT-cell activation response about equal to or less than that of hB7-1.

Also included are isolated or recombinant polypeptides comprising anamino acid sequence having at least about 75% sequence identity to atleast one of SEQ ID NOS:263–272, or a subsequence thereof comprising theextracellular domain, where the amino acid sequence is notnaturally-occurring, and the polypeptide has a CTLA-4/CD28 bindingaffinity ratio about equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of human B7-1, and/or an ability to induce a T-cellproliferation and/or activation response about equal to or less thanthat of hB7-1.

In yet another aspect, the invention includes an isolated or recombinantpolypeptides which comprises a non naturally-occurring amino acidsequence encoded by a nucleic acid comprising a polynucleotide sequenceselected from: (a) a polynucleotide sequence selected from SEQ IDNOS:22–45, 143–173, 253–262, or a complementary polynucleotide sequencethereof; (b) a polynucleotide sequence encoding a polypeptide selectedfrom SEQ I) NOS:69–92, 222–247, 263–272, 286–289, or a complementarypolynucleotide sequence thereof; (c) a polynucleotide sequence which,but for the degeneracy of the genetic code, hybridizes under highlystringent conditions over substantially the entire length ofpolynucleotide sequence (a) or (b); (d) a polynucleotide sequencecomprising all or a fragment of (a), (b), or (c), wherein the fragmentencodes a polypeptide having a CTLA-4/CD28 binding affinity ratio aboutequal to or greater than the CTLA-4/CD28 binding affinity ratio of humanB7-1, or an ability to induce a T-cell proliferation or activationresponse about equal to or less than that of hB7-1; (e) a polynucleotidesequence encoding a polypeptide, the polypeptide comprising an aminoacid sequence which is substantially identical over at least about 150contiguous amino acid residues of any one of SEQ ID NOS:69–92, 222–247,263–272, 286–289, and (f) a polynucleotide sequence encoding apolypeptide that has a CTLA-4/CD28 binding affinity ratio about equal toor greater than the CTLA-4/CD28 binding affinity ratio of human B7-1 oran ability to induce a T-cell proliferation or activation response aboutequal to or less than that of hB7-1, which polynucleotide sequence hasat least about 70% identity to at least one polynucleotide sequence of(a), (b), (c), or (d).

The invention also includes an isolated or recombinant polypeptidecomprising an amino acid sequence according to the formula:

MGHTRRQGTSP-X12-KCPYLKFFQLLV-X25-ACL-X29-HLCSGVIHVT-X40-EVKEVATLSCGLNVSVEELAQTRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKY-X122-KDAFKR-X129-HLAEVMLSVKADFPTPSITDFEIPPSNIRRIICS-X164-SGGFPEPHLFWLENGEELNAINTTVSQDPET-X196-LYTVSSKLDFNMTANHSFMCLI-X219-YGHLRVNQTFNWNTPKQEHFP-X241-NLLPSWAITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV (SEQ ID NO:287), or asubsequence thereof comprising the extracellular domain, whereinposition X12 is Ser or Pro; position X25 is Leu or Met; position X29 isSer or Pro; position X40 is Lys or Arg; position X122 is Glu or Asp;position X129 is Glu or Lys; position X164 is Thr or Ala; position X196is Glu or Gly; position X219 is Lys or Arg; position X241 is Asp or Asn.Some such polypeptides have a CTLA-4/CD28 binding affinity ratio aboutequal to or greater than the CTLA-4/CD28 binding affinity ratio ofhB7-1, and/or ability to induce T-cell proliferation or activation orboth about equal to or less than that induced by hB7-1.

The invention also provides an isolated or recombinant polypeptidecomprising a subsequence of an amino acid sequence set forth in any ofSEQ ID NOS:69–92, 222–247, 263–272, and 286–289, wherein the subsequenceis the extracellular domain or full-length sequence of such amino acidsequence. Furthermore, the invention includes the full-lengthpolypeptide sequence and one or more subsequences thereof, e.g., signalpeptide, extracellular domain (ECD), transmembrane domain (TMD), and/orcytoplasmic domain (CD) of any of SEQ ID NOS:66, 81, 85, 86, 88, 90, 91,285, 288, 289, 291, and 294, and nucleic acid sequences encoding any ofthese amino acid sequences.

The invention provides isolated or recombinant nucleic acids comprisinga polynucleotide sequence selected from: (a) a polynucleotide sequenceselected from SEQ ID NOS:1–21 and 95–142, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:48–68, 174–221, 283–285, and290–293, or a complementary polynucleotide sequence thereof; (c) apolynucleotide sequence which, but for codon degeneracy, hybridizesunder at least stringent or highly stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b);and (d) a polynucleotide sequence comprising all or a nucleotidefragment of (a), (b), or (c), wherein the fragment encodes a polypeptidehaving a CD28/CTLA-4 binding affinity ratio about equal to or greaterthan the CD28/CTLA-4 binding affinity ratio of human B7-1, or an abilityto induce a T-cell proliferation or activation response about equal toor greater than that of hB7-1. In some such nucleic acids, thepolynucleotide sequence of (d) encodes a nucleotide fragment of (a) or(b) that encodes an ECD having a CD28/CTLA-4 binding affinity ratio oran ability to induce a T-cell proliferation or activation response aboutequal to or greater than that of hB7-1.

The invention also includes isolated or recombinant nucleic acidscomprising a polynucleotide sequence encoding a polypeptide, wherein theencoded polypeptide comprises an amino acid sequence which is (a)substantially identical over at least about 150 or 200 contiguous aminoacid residues of any one of SEQ ID NOS:48–68, 174–221, 283–285, and290–293 and (b) is a non naturally-occurring sequence.

In addition, the invention includes isolated or recombinant nucleicacids comprising a nucleotide sequence coding for a polypeptidecomprising the amino acid sequence set forth in any of SEQ ID NOS:48–68,174–221, 283–285, and 290–293, or a subsequence thereof, wherein thesubsequence comprises at least one of: the signal sequence,extracellular domain, or transmembrane domain of said polypeptide, andthe cytoplasmic domain of said polypeptide, and wherein the amino acidsequence or subsequence is a non naturally-occurring sequence.Similarly, fragments of the above nucleotides that encode a polypeptidethat has a substantially equivalent or equivalent binding activity of aNCSM polypeptide molecule, produces a substantially equivalent orequivalent NCSM-polypeptide-mediated immune response, e.g., induction orinhibition of T cell activation or proliferation, or cytokine productionare a feature.

Also provided is an isolated or recombinant nucleic acid comprising apolynucleotide sequence selected from: (a) a polynucleotide sequenceselected from SEQ ID NOS:22–45, 143–173, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:69–92, 222–247, 286–289, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which, but for the degeneracy of the genetic code, hybridizesunder highly stringent conditions over substantially the entire lengthof polynucleotide sequence (a) or (b); and (d) a polynucleotide sequencecomprising all or a fragment of (a), (b), or (c); wherein (c) or (d)encodes a polypeptide having a non naturally-occurring sequencecomprising at least one of:

Gly at position 2; Thr at position 4; Arg at position 5; Gly at position8; Pro at position 12; Met at position 25; Cys at position 27; Pro atposition 29; Leu at position 31; Arg at position 40; Leu at position 52;His at position 65; Ser at position 78; Asp at position 80; Tyr atposition 87; Lys at position 120; Asp at position 122; Lys at position129; Met at position 135; Phe at position 150; Ile at position 160; Alaat position 164; His at position 172; Phe at position 174; Leu atposition 176; Asn at position 178; Asn at position 186; Glu at position194; Gly at position 196; Thr at position 199; Ala at position 210; Hisat position 212; Arg at position 219; Pro at position 234; Asn atposition 241; Leu at position 244; Thr at position 250; Ala at position254; Tyr at position 265; Arg at position 266; Glu at position 273; Lysat position 275; Ser at position 276; an amino acid deletion at position276; and Thr at position 279, wherein the position number corresponds tothat of the human B7-1 amino acid sequence (SEQ ID NO:278), and whereinsaid polypeptide has a CTLA-4/CD28 binding affinity ratio about equal toor greater than the CTLA-4/CD28 binding affinity ratio of human B7-1.

The invention further provides an isolated or recombinant nucleic acidcomprising a polynucleotide sequence selected from: (a) a polynucleotidesequence selected from SEQ ID NOS:253–262, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:263–272, or a complementarypolynucleotide sequence thereof; (c) a polynucleotide sequence which,but for codon degeneracy, hybridizes under highly stringent conditionsover substantially the entire length of polynucleotide sequence (a) or(b) and encodes a polypeptide having a non naturally-occurring sequence;and (d) a polynucleotide sequence comprising all or a fragment of (a),(b), or (c), wherein the fragment encodes a polypeptide having (i) a nonnaturally-occurring sequence and (ii) a CTLA-4/CD28 binding affinityratio about equal to or greater than the CTLA-4/CD28 binding affinityratio of human B7-1, or an ability to induce a T-cell proliferation oractivation response about equal to or less than that of hB7-1.

The invention also includes an isolated or recombinant nucleic acidcomprising a polynucleotide sequence encoding a polypeptide thatcomprises an amino acid sequence which is substantially identical overat least about 150 contiguous amino acid residues of any one of SEQ IDNOS:69–92, 222–247, 263–272, and 286–289.

The invention also provides an isolated or recombinant nucleic acidcomprising a nucleotide sequence coding for a polypeptide comprising theamino acid sequence set forth in any of SEQ ID NOS:69–92, 222–247,263–272, and 286–289, or a subsequence thereof, wherein the subsequencecomprises at least one of the signal sequence, ECD, transmembranedomain, and cytoplasmic domain of said polypeptide, and the amino acidsequence or subsequence is a non naturally-occurring sequence.

In another aspect, the invention provides an isolated or recombinantnucleic acid encoding a polypeptide that has a CTLA-4/CD28 bindingaffinity ratio about equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of human B7-1, or an ability to induce a T-cellproliferation and/or activation response about equal to or greater thanthat of hB7-1, produced by mutating or recombining at least one nucleicacids described above. Also included is an isolated or recombinantpolypeptide comprising an amino acid sequence having the formula:

MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPK-SVTKRVKETVM-X50-SCDY-X55-X56-STEELTSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITD-MNDNPRIVILALRLSD-X113-GTYTCV-X120-QK-X123-X124-X125-X126-G-X128-X129-X130-X131-EHL-X135-SV-X138-L-X140-IRADFPVPSITDIGHPAPNVKRIRCSASG-X170-FPEPRLAWMEDGEEL-NAVNTTV-X193-X194-X195-LDTELYSVSSELD-X209-N-X211-TNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVI-X252-X253-VSGALVLTAVVLYCLACRHVAR (SEQ ID NO:290), or asubsequence thereof comprising the extracellular domain, whereinposition X50 is Leu or Pro; position X55 is Asn or Ser; position X56 isAla or Thr; position X113 is Ser or Lys; position X120 is Ile or Val;position X123 is Pro or deleted; position X124 is Val, Asn, or Asp;position X125 is Leu or Glu; position X126 is Lys or Asn; position X128is Ala or Ser; position X129 is Tyr or Phe; position X130 is Lys or Arg;position X131 is Leu or Arg; position X135 is Ala or Thr; position X138is Arg or Thr; position X140 is Met or Ser; position X170 is Asp or Gly;position X193 is Asp or is deleted; position X194 is Gln or is deleted;position X195 is Asp or is deleted; position X211 is Val or Ala;position X252 is Ile or Val; and position X253 is Leu or Pro. Thepolypeptide may comprise a sequence of any of SEQ ID NOS:59, 62, 180,184, 188, 195, 196, 200, 201, 204, 211, 213, 219, and 291. Some suchpolypeptides have a CD28/CTLA-4 binding affinity ratio about equal to orgreater than that of hB7-1, and/or an ability to induce T-cellproliferation and/or activation about equal to or greater than thatinduced by hB7-1.

Another feature of the invention is an isolated or recombinantpolypeptide comprising an amino acid sequence according to the formula:

MGHTMKWG-X9-LPPKRPCLWLSQLLVLTGLFYFCSG-X35-TPKSVTKRVKETVMLSCDY-X55-TSTEELTSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALR-X110-SDSGTYTCVIQKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENG-X183-ELNATNTT-X192-SQDPETKLYMISSELDFN-X211-TSN-X215-X216-X217-LCLVKYGDLTVSQ-X231-FYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEV-X288-M-X290-SCSQSP (SEQ ID NO:292), or asubsequence thereof comprising the extracellular domain, whereinposition X9 is Thr or Ser; position X35 is Ile or Thr; position X55 isAsn or Ser; position X110 is Leu or Pro; position X124 is Asp or Val;position X135 is Thr or Ala; position X183 is Lys or Glu; position X192is Leu or Val; position X211 is Met or Thr; position X215 is His or isdeleted; position X216 is Ser or is deleted; position X217 is Phe or isdeleted; position X231 is Thr or Ser; position X288 is Lys or Glu;position X290 is Glu or Gln, and wherein said amino acid sequence is anon naturally-occurring sequence. Some such polypeptides have aCD28/CTLA-4 binding affinity ratio about equal to or greater than thatof hB7-1, and/or an ability to induce T-cell proliferation and/oractivation about equal to or greater than that induced by hB7-1.

The invention includes an isolated or recombinant polypeptide comprisingthe amino acid sequence of SEQ ID NO:93 or SEQ ID NO:94, or asubsequence thereof, wherein the subsequence comprises at least one ofthe signal sequence, ECD, transmembrane domain, and cytoplasmic domainof said polypeptide. Also provided is an isolated or recombinant nucleicacid comprising a polynucleotide sequence selected from: (a) apolynucleotide sequence selected from SEQ ID NO:46 or SEQ ID NO:47, or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence encoding a polypeptide selected from SEQ ID NO:93, SEQ IDNO:94, or a complementary polynucleotide sequence thereof; (c) apolynucleotide sequence encoding a subsequence of a polypeptide selectedfrom SEQ ID NO:93, SEQ ID NO:94, or a complementary polynucleotidesequence thereof, wherein the subsequence comprises at least one of: thesignal sequence, extracellular domain, transmembrane domain, andcytoplasmic domain of the polypeptide.

In another aspect, the invention provides a polypeptide which isspecifically bound by a polyclonal antisera raised against at least oneantigen, the antigen comprising the polypeptide sequence selected fromany of SEQ ID NOS:48–94, 174–252, 263–272, 283–293, or a fragmentthereof, wherein the antisera is subtracted with one or more (andoptionally all) polypeptides encoded by one or more of the sequences setforth at GenBank Nucleotide Accession Nos: A92749, A92750, AA983817,AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894,AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829,AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443,AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843,L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191,S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832,U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. Some suchpolypeptides have a CTLA-4/CD28 binding affinity ratio about equal to orgreater than the CTLA-4/CD28 binding affinity ratio of human B7-1,and/or an ability to induce a T-cell proliferation or T-cell activationresponse about equal to or greater than that of hB7-1.

The invention further includes an antibody or antisera produced byadministering any NCSM polypeptide described above to a mammal, whichantibody specifically binds at least one antigen, the antigen comprisinga polypeptide comprising at least one amino acid sequence of any of SEQID NOS:48–94, 174–252, 263–272, and 283–293, which antibody does notspecifically bind to a polypeptide encoded by at least one (optionallyall) of the sequences at GenBank Nucleotide Accession Nos: A92749,A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465,AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825,AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834,AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904,AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075,M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925,U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823,and Y09950.

The invention provides an antibody or antisera which specifically bindsa polypeptide which comprises any sequence selected from any of SEQ IDNOS:48–94, 174–252, 263–272, and 283–293, wherein the antibody orantisera does not specifically bind to at least one (optionally all)polypeptide encoded by at least one of GenBank Nucleotide Accession Nos:A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153,AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824,AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833,AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762,AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074,M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593,U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958,Y08823, and Y09950. The antibodies are, e.g., polyclonal, monoclonal,chimeric, humanized, single chain, Fab fragments, fragments produced byan Fab expression library, or the like.

In another aspect, the invention provides a nucleic acid which comprisesa unique subsequence in a nucleic acid selected from SEQ ID NOS:1–47,95–173, and 253–262, wherein the unique subsequence is unique ascompared to at least one (optionally all) nucleic acid corresponding toany of GenBank Nucleotide Accession Nos.: A92749, A92750, AA983817,AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894,AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829,AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443,AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843,L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191,S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832,U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. Theinvention also includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from: SEQ ID NOS:48–94, 174–252,263–272, and 283–293, wherein the unique subsequence is unique ascompared to at least one (optionally all) polypeptide encoded by any ofGenBank Nucleotide Accession Nos. shown above.

The invention includes a target nucleic acid which, but for nucleotidecodon degeneracy, hybridizes under stringent conditions to a uniquecoding oligonucleotide that encodes a unique subsequence in apolypeptide selected from SEQ ID NOS:48–94, 174–252, 263–272, and283–293, wherein the unique subsequence is unique as compared to atleast one (optionally all) polypeptide encoded by any of GenBankNucleot. Access. Nos.: A92749, A92750, AA983817, AB026121, AB030650,AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896,AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831,AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653,AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533,M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695,S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755,U88622, X60958, Y08823, and Y09950.

The invention also includes compositions comprising any polypeptideand/or polynucleotide described herein in an excipient, preferably apharmaceutically acceptable excipient. In one aspect, the inventionprovides compositions comprising an isolated or recombinant NCSMpolypeptide comprising the amino acid sequence SEQ ID NOS:48–68,174–221, 283–285, 290–293, or a costimulatory fragment thereof, whereinsaid costimulatory fragment has a CD28/CTLA-4 binding affinity ratioabout equal to or greater than the CD28/CTLA-4 binding affinity ratio ofhuman B7-1, or an ability to induce a T-cell proliferation or activationresponse about equal to or greater than that of hB7-1, and a carrier orexcipient. Compositions comprising an isolated or recombinant NCSMpolypeptide comprising the amino acid sequence of SEQ ID NOS:69–92,222–247, 263–272, 286–289, or a costimulatory fragment thereof, whereinsaid costimulatory fragment has a CTLA-4/CD28 binding affinity ratioabout equal to or greater than the CTLA-4/CD28 binding affinity ratio ofhuman B7-1, or an ability to induce a T-cell proliferation or activationresponse about equal to or less than that of hB7-1, and a carrier arealso a feature of the invention.

Also included are isolated or recombinant polypeptides, each comprisingan amino acid sequence corresponding to an extracellular domain, whereinsaid amino acid sequence has at least about 92%, 93%, 94%, 95% or moreamino acid sequence identity to the amino acid sequence corresponding tothe extracellular domain of SEQ ID NO:66, and wherein said polypeptidehas a CD28/CTLA-4 binding affinity ratio greater than the CD28/CTLA-4binding affinity ratio of human B7-1. Some such polypeptides may furthercomprise at least one further amino acid sequence corresponding to asignal peptide, transmembrane domain or a cytoplasmic domain.

The invention also includes isolated or recombinant polypeptidevariants, each of which comprises an amino acid sequence that differsfrom the amino acid sequence of a primate B7-1, wherein the differencebetween the amino acid sequence of the variant and the amino acidsequence of the primate B7-1 comprises a different amino acid atposition 65 other than alanine, wherein the position corresponds to theposition in the amino acid sequence of human B7-1 of SEQ ID NO:278. Thedifferent amino acid may comprise His, Arg, Lys, Pro, Phe, or Trp, andthe primate B7-1 may be hB7-1. Some such polypeptide variants have aCTLA-4/CD28 binding affinity ratio greater than that or hB7-1 or anability to induce a T cell proliferation response less than that ofhB7-1.

The invention also includes an isolated or recombinant nucleic acidcomprising a polynucleotide sequence encoding a polypeptide, where thepolypeptide comprises an amino acid sequence which is substantiallyidentical over at least 175 contiguous amino acids of any one of thoseNCSM polypeptide sequences listed. In various embodiments, the encodedpolypeptide comprises at least about 100, 150, 170, 180, 190, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 225, 230, 240, 250,260, 265, 270, 275, or 285 or more contiguous amino acid residues orsubstantially identical variants of any one of the polypeptide sequenceslisted, or encoded by any nucleic acid listed. These polypeptides canexist separately or as components of one of more fusion proteins.

The invention also includes a cell comprising any nucleic acid describedherein, or which expresses any polypeptide or nucleic acid noted herein.In one embodiment, the cell expresses a polypeptide encoded by thenucleic acids herein.

The invention also includes a vector comprising any nucleic acid of theinvention. The vector can comprise a plasmid, a cosmid, a phage, or avirus or a virus-like particle (VLP) (or virus fragment); the vector canbe, e.g., an expression vector, a cloning vector, a packaging vector, anintegration vector, or the like. The invention also includes a celltransduced by the vector. The invention also includes compositionscomprising any nucleic acid described herein, and an excipient,preferably a pharmaceutically acceptable excipient. Cells and transgenicanimals that include any polypeptide or nucleic acid herein, e.g.,produced by transduction of the vector, are also a feature of theinvention.

The invention also includes compositions produced by digesting one ormore of the nucleic acids described herein with a restrictionendonuclease, an RNAse, or a DNAse; and, compositions produced byincubating one or more nucleic acids described herein in the presence ofdeoxyribonucleotide triphosphates and a nucleic acid polymerase, e.g., athermostable polymerase.

The invention also includes compositions comprising two or more nucleicacids described herein. The composition may comprise a library ofnucleic acids, where the library contains at least 5, 10, 20 or 50 ormore nucleic acids.

In another aspect, the invention includes an isolated or recombinantpolypeptide encoded by any nucleic acid described herein. In oneembodiment, the polypeptide may comprise a sequence selected from any ofSEQ ID NOS:48–94, 174–252, 263–272, and 283–293. These sequences andfragments thereof can be present separately or as components of largerproteins such as fusion proteins.

Any polypeptide described herein optionally can effect or alter animmune response, e.g., either induce or inhibit proliferation oractivation of T cells. In other embodiments, any polypeptide describedabove can bind preferentially either CD28 or CTLA-4 or both CD28 andCTLA-4 as described herein. In other embodiments, any polypeptidedescribed herein optionally can enhance or limit cytokine production asdescribed herein. Nucleotides encoding any such polypeptides havingthese properties are also a feature of the invention.

In one class of embodiments, any polypeptide described herein mayfurther include a secretion signal or localization signal sequence.e.g., a signal sequence, an organelle targeting sequence, a membranelocalization sequence, and the like. Any polypeptide described hereinmay further include a sequence that facilitates purification, e.g., anepitope tag (such as, e.g., a FLAG™ epitope), a polyhistidine tag, a GSTfusion, and the like. The polypeptide optionally includes a methionineat the N-terminus. Any polypeptide described herein optionally includesone or more modified amino acids, such as a glycosylated amino acid, aPEG-ylated amino acid, a farnesylated ammo acid, an acetylated aminoacid, a biotinylated amino acid, a carboxylated amino acid, aphosphorylated amino acid, an acylated amino acid, or the like. Anypolypeptide described herein further may be incorporated into a fusionprotein, e.g., a fusion with an immunoglobulin (Ig) sequence.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any NCSM nucleic acid described herein, which is operativelylinked to a regulatory sequence effective to produce the encodedpolypeptide, culturing the cells in a culture medium to produce thepolypeptide, and isolating the polypeptide from the cells or from theculture medium. Another such method comprises introducing into apopulation of cells a recombinant expression vector comprising any NCSMnucleic acid described herein; administering the expression vector intoa mammal; and isolating the polypeptide from the mammal or from abyproduct of the mammal.

The invention also includes a method of treating an autoimmune orallergic disorder in a subject in need of such treatment byadministering to the subject an effective amount of any NCSM polypeptide(or polynucleotide or expression vector encoding such polypeptide)described herein. In various embodiments, the autoimmune disorder may bemultiple sclerosis, rheumatoid arthritis, lupus erythematosus, type Idiabetes, psoriasis and the like.

The invention also includes a method of enhancing or reducing an immuneresponse in a subject, such as either by inducing or inhibiting T cellproliferation or activation, by administration of at least one NCSMpolypeptide and/or NCSM polynucleotide described herein to a populationof cells. The population of cells to which the nucleic acid orpolypeptide is administered can be in vivo, ex vivo, or in vitro (e.g.,cultured cells). The invention includes a method of inducing, modifying,or inhibiting T-cell proliferation, the method comprising contacting apopulation of T cells with a polypeptide of the invention, therebyinducing, modifying, or inhibiting, respectively, proliferation of the Tcells (relative to the response generated by WT hB7-1). Polypeptidesthat induce such T cell proliferation include CD28BP polypeptides;polypeptides that inhibit T cell proliferation include the CTLA-4BPpolypeptides and B7-1 and B7-2 polypeptide variants discussed herein.

The invention also includes, in a method of treating a disorder ormedical condition treatable by administration of NCSM polypeptides (orfragments thereof) or NCSM polynucleotides (or fragments thereof) to asubject, an improvement comprising administering to the subject aneffective amount of a polypeptide and/or nucleic acid (or fragmentsthereof) described herein. The disorder, disease, or medical conditiontreatable by administration of NCSM polypeptides and/or nucleic acids(or fragments thereof, including soluble NCSMs and fusion proteins andvectors encoding them) may be, but is not limited to, e.g., chronicdisease, autoimmune disorder, multiple sclerosis, rheumatoid arthritis,lupus erythematosus, type I diabetes, psoriasis, AIDS or AIDS-relatedcomplexes, allogeneic or xenogeneic grafts or transplants, a variety ofcancers, viral and/or bacterial infections, or the like.

Also included is a method of therapeutic or prophylactic treatment of adisease or disorder in a subject in need of such treatment, comprisingadministering to the subject any NCSM polypeptide described herein andan immunogen specific for said disease or disorder, wherein the combinedamount of polypeptide and immunogen is effective to prophylactically ortherapeutically treat said disease or disorder.

In yet another aspect, the invention includes a method of enhancing,diminishing, modifying, or potentiating an immune response in a subject,comprising: directly administering to the subject a polynucleotidecomprising any NCSM nucleic acid sequence described herein, operablylinked to a promoter sequence that controls the expression of saidnucleic acid sequence, said polynucleotide being present in an amountsufficient that uptake of said polynucleotide into one or more cells ofthe subject occurs and sufficient expression of said nucleic acidsequence results to produce an amount of a polypeptide effective toenhance, diminish, or modify an immune response.

In another aspect, the invention provides a method of modulating oraltering a T-cell response specific to an antigen in a subject, themethod comprising administering to the subject at least onepolynucleotide sequence encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272 and 283–293 or fragment thereof, and apolynucleotide sequence encoding the antigen or antigenic fragmentthereof, wherein each of the at least one polynucleotide sequences isexpressed in the subject in an amount effective to modulate or alter a Tcell response.

The invention also includes a method of modulating or altering an immuneresponse in a subject, the method comprising introducing into cells of atumor of the subject at least one polynucleotide sequence encoding apolypeptide comprising any of SEQ ID NOS:48–94, 174–252, 263–272 and283–293 or fragment thereof, wherein the polypeptide or fragment thereofinteracts with or binds to a T cell receptor when expressed in asubject, and wherein the at least one polynucleotide sequence isoperably linked to a promoter for expression in the subject and ispresent in an amount sufficient that when expressed is effective tomodulate or alter a T cell response.

In addition, the invention includes a vector comprising at least onepolynucleotide sequence encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272 and 283–293 or fragment thereof, wherein thepolypeptide or fragment thereof interacts with or binds to a T cellreceptor when expressed in a subject, wherein the at least onepolynucleotide sequence is operably linked to a promoter for expressionin the subject and is present in an amount sufficient that whenexpressed is effective to modulate or alter a T cell response.

In another aspect, the invention provides vector comprising at least onepolynucleotide sequence encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272 and 283–293 or fragment thereof, and apolynucleotide sequence encoding the antigen or antigenic fragmentthereof, wherein the NCSM polypeptide or fragment thereof interacts withor binds to a T cell receptor when expressed in a subject, and whereineach of the at least one polynucleotide sequences is operably linked toa promoter for expression in the subject and is present in an amountsufficient that when expressed is effective to modulate or alter a Tcell response.

In general, nucleic acids and proteins derived by mutation, recursivesequence recombination (RSR) or other alterations of the sequencesherein are a feature of the invention. Similarly, those produced byrecombination, including recursive sequence recombination, are a featureof the invention. Mutation and recombination methods using the nucleicacids described herein are a feature of the invention. For example, onemethod of the invention includes recombining one or more nucleic acidsdescribed herein with one or more additional nucleic acids (including,but not limited to those noted herein), the additional nucleic acidencoding a NCSM polypeptide, co-stimulatory homologue or subsequencethereof. The recombining steps are optionally performed in vivo, exvivo, or in vitro. Also included in the invention are a recombinantnucleic acid produced by this method, a cell containing the recombinantnucleic acid, a nucleic acid library produced by this method comprisingrecombinant polynucleotides, and a population of cells containing thelibrary comprising recombinant polynucleotides.

The invention also includes a method of designing or identifyingagonists and antagonists of CD28 and CTLA-4 (which either enhance orinhibit signaling through CD28 or CTLA-4) based on the 3-dimensionalstructure of the polypeptides of the invention (e.g., SEQ ID NOS:48–94,174–252, 263–272, and 283–293).

The invention also includes soluble polypeptides and proteins (includingfusion polypeptides and proteins) and nucleic acids encoding suchsoluble polypeptides and proteins. The invention also includes the useof such polypeptides and proteins as therapeutics, prophylactics, anddiagnostics in therapeutic treatment and/or prevention of a variety ofdiseases and conditions. Soluble polypeptides and proteins (and nucleicacids encoding them) include, e.g., extracellular domain (ECD) aminoacid sequences of each NCSM (e.g., each CTLA-4 binding protein and CD28binding protein) described herein (or fragments thereof) and nucleicacids encoding same, as well as constructs comprising, e.g., each ofsaid ECD, or fragments thereof, with an Ig polypeptide sequence (orfragment or variant thereof) (and nucleotide sequences encoding same) asfusion proteins. Some such soluble polypeptides and proteins exhibit aCD28/CTLA-4 binding affinity ratio that is greater than that of hB7-1and/or have an ability to induce a T cell proliferation response, in thepresence of activated T cells, that is less than that induced by solublehB7-1 polypeptide in the presence of activate T cells.

In another aspect, the invention provides a computer or computerreadable medium comprising a database comprising a sequence recordcomprising one or more character strings corresponding to a nucleic acidor protein sequence selected from any of SEQ ID NOS:1–272 and 283–293.The invention further includes an integrated system comprising acomputer or computer readable medium comprising a database comprisingone or more sequence records, each comprising one or more characterstrings corresponding to a nucleic acid or protein sequence selectedfrom any of SEQ ID NOS:1–272 and 283–293, the integrated system furthercomprising a user input interface allowing a user to selectively viewone or more sequence records. Also provided are methods of using acomputer system to present information pertaining to at least one of aplurality of sequence records stored in a database, said sequencerecords each comprising one or more character strings corresponding toany of SEQ ID NOS:1–272 and 283–293.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of the following exemplaryinteractions: 1) interactions between a T cell receptor (TCR) andantigenic peptide presented in the groove of a major histocompatibilitycomplex (MHC) molecule, and 2) interactions between a recombinant CD28BPpolypeptide of the invention expressed on the surface of anantigen-presenting cell (APC) and a CD28 receptor on a T cell. FIG. 1Bis a schematic representation of exemplary interactions: 1) between aTCR and antigenic peptide presented in the groove of a MHC molecule, and2) between a recombinant CTL4-BP polypeptide of the invention expressedon the surface of an APC and a CTLA-4 receptor on a T cell. Therepresentation illustrates the principle by which recombinantpolypeptides of the invention which preferentially bind the CD28 orCTLA-4 receptor effectuate enhanced or suppressed T cell activation.

FIGS. 2A–2H depict an alignment of a naturally-occurring (i.e.,wild-type) human B7-1 polypeptide sequence (SEQ ID NO:278) and exemplaryCD28BP polypeptide sequences of the invention (SEQ ID NOS:48–68, SEQ IDNOS:174–221, and SEQ ID NO:283). The predicted boundaries between thesignal peptide region, extracellular domain (ECD), transmembrane domain(TMD), and cytoplasmic domain (CD), based on corresponding boundaries inthe hB7-1 sequence are shown at the top. SEQ ID NO:283 represents a“consensus sequence” of these aligned CD28BP sequences. The arrowpositioned between the amino acid residues equivalent to amino acidresidues 34–35 of SEQ ID NO:278, indicates the predicted boundarybetween the signal peptide region and the mature polypeptide regionbased on comparison with the hB7-1 sequence.

FIGS. 3A–3H illustrate an alignment of a naturally-occurring (i.e.,wild-type) hB7-1 polypeptide sequence (SEQ ID NO: 278) and exemplaryCTLA-4BP polypeptide sequences of the invention (SEQ ID NOS: 69–73, SEQID NOS:74–92, SEQ ID NOS:222–252, and SEQ ID NO:286). The predictedboundaries between the signal peptide sequence, ECD, TMD, and CD, basedon corresponding boundaries in the hB7-1 sequence are shown at the top.

SEQ ID NO:286 represents a “consensus sequence” of these alignedCTLA-4BP sequences of the invention. Alignments shown in FIGS. 2A–2H and3A–3H were prepared using the CLUSTALW multiple sequence alignmentprogram, a part of the Vector NTI version 6 sequence analysis softwarepackage (Informax, Bethesda, Md.). CLUSTALW initially performs multiplepairwise comparisons between groups of sequences and then assembles thepairwise alignments into a multiple alignment based on homology. For theinitial pairwise alignments, Gap Open and Gap Extension penalties were10 and 0.1, respectively. For the multiple alignments, Gap Open penaltywas 10, and the Gap Extension penalty was 0.05. The BLOSUM62 matrix wasthe protein weight matrix.

FIGS. 4A–4D presents graphs illustrating competitive FACS bindingprofiles of hB7-1, clone CD28BP-15, clone CTLA-4BP 5x4-12c, and vectorcontrol for each of soluble CD28-Ig receptor and soluble CTLA-4-Igreceptor.

FIG. 5 presents graphs depicting competitive FACS binding profiles ofseventeen Round 2 CD28BP clones for each of soluble CD28-Ig receptor andsoluble CTLA-4-Ig receptor.

FIG. 6A is a schematic representation of an exemplary competitive FACSbinding profile for a CTLA-4BP clone for soluble CD28-Ig receptor andsoluble CTLA-4-Ig receptor. FIG. 6B is a schematic representation of anexemplary competitive FACS binding profile for a CD284BP clone forsoluble CD28-Ig receptor and soluble CTLA-4-Ig receptor.

FIGS. 7A–7H are graphs showing competitive FACS binding profiles of WThuman B7-1 (CD80), five CTLA-4BP clones, and HEK 293 cells (control) forsoluble CD28-Ig receptor and soluble CTLA-4-Ig receptor.

FIGS. 8A–8B present schematic representations of the amino acidsequences of CD28BP-15 and CTLA-4BP 5x4-12c and the genealogy of thesesequences.

FIGS. 9A–9F are graphs depicting the mean fluorescence intensitiesgenerated by the binding of labeled soluble ligand sCD28-Ig and labeledsoluble ligand sCTLA-4-Ig to clones CD28BP-15 and CTLA-4BP 5x4-12c.FIGS. 9G–9H provide graphs illustrating histograms from the staining ofstable 293 transfectants expressing CTLA-4BP 5x4-12c (gray histograms),hB7-1 (gray histograms) and negative control transfectants (openhistograms) with anti-hB7-1 monoclonal antibodies (mAbs) with expressionlevels analyzed by flow cytometry.

FIG. 10 shows a graph depicting T cell proliferation response, asmeasured by ³H thymidine incorporation, resulting from the co-culturingof cells transfected with one of seventeen CD28BP clones, human B7-1(CD80), or an empty control vector cultured with anti-CD3 mAbs.

FIGS. 11A–11C present graphs illustrating improved co-stimulation ofpurified human T cells observed co-culturing irradiated 293 cellstransiently (A) or stably (B) transfected with clone CD28BP-15, hB7-1,or a control vector with purified T cells and anti-CD3 mAbs. FIG. 11Cshows a graph depicting levels of IFN-gamma produced by co-culturingirradiated stable transfectants expressing CD28BP or hB7-1 or negativecontrol cells transfected with an “empty” vector with purified human Tcells.

FIG. 12 shows a graph depicting T cell proliferation response, asmeasured by ³H thymidine incorporation, resulting from the co-culturingof cells transfected with one of nineteen CTLA-4BP clones, WT human B7-1(CD80), or an empty control vector cultured with soluble anti-CD3 mAbs.

FIGS. 13A–13D show graphs illustrating the effects of cells transfectedwith clone CTLA-4 BP 5x4-12C, hB7-1 or a control vector cultured on Tcell proliferation induced by co-culturing the transfectants withpurified T cells in the presence of soluble anti-CD3 mAbs and oncytokine synthesis in mixed lymphocyte reaction assay.

FIGS. 14A–14B are schematic representations of exemplary soluble formsof human B7-1 molecules. Expression plasmids were constructed byjuxtaposing the nucleotide sequence encoding a signal sequence andextracellular domain (ECD) (or ECD fragment) of WT hB7-1 with anucleotide sequence encoding E epitope and/or His Tag or human Ig Fcdomain to create a IgG fusion protein. FIG. 14A shows a representationof a fusion protein expressed by one such plasmid comprising a solubleWT human B7-1-ECD, including a signal sequence peptide (amino acidresidues 1–34), ECD (amino acid residues 35–242), and E-epitope tag(amino acid residues 243–259) and His-tag (amino acid residues 260–268).Numbering coincides with the ATG or Met. The amino acid residuespositioned at the beginning and end of an exemplary ECD amino acidsequence are shown. FIG. 14B is an illustration of a fusion proteinexpressed by one such plasmid comprising a soluble WT hB7-1-ECD-Igfusion protein, including the signal domain (amino acid residues 1–34),ECD domain (amino acid residues 35–242), Factor Xa (IGER),valine-threonine (VT) or (BsetII) glycine-valine-threonine (GVT) linker,and hinge CH2-CH3 (constant/heavy) region of the Fc domain of IgG1(e.g., GenBank Access. No. P01857 or X70421) (showing the initial aminoacid residues corresponding to nucleic acid sequence shown at GenBankAccession No. X70421). The amino acid residues positioned at thebeginning and end of an exemplary ECD amino acid sequence are shown.Optionally, other Ig molecules, or Ig Fc fragments thereof, can used toconstruct NCSM-Ig fusion proteins. Similar expression plasmids wereconstructed by substituting a nucleotide sequence encoding a NCSMpolypeptide of the invention for the sequence encoding the hB7-1 ECDdomain, and fusion proteins comprising NCSM-ECD sequences were generatedfrom such plasmids. A nucleotide sequence encoding truncated ECD domainof hB7-1 or a NCSM polypeptide can also be substituted. The signalsequence may be the WT hB7-1 signal sequence or a recombinant signalsequence from a recombinant NCSM polynucleotide. The B7-1- orNCSM-ECD-Ig fusion protein may include Factor Xa cleavage site.

FIG. 15 illustrates an example of a pNCSMsECD plasmid expression vectorcomprising a nucleotide sequence encoding a soluble extracellular domainof a NCSM polypeptide of the invention with an E-epitope tag and/orhistidine tag.

FIG. 16 is a photographic representation of a sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of varioussoluble forms of WT B7-1 (ECD and fusion protein and delta Cys mutant)and clone CD28BP-15. Molecular weight standards are shown on the leftfor comparison: myosin (185 kDa); phosphorylase B (98 kDa); glutamicdehydrogenase (52 kDa); carbonic anhydrase (31 kDa); myoglobin (19 kDa);and lysozyme (11 kDa).

FIG. 17 illustrates an example of a phB7-1ECD-Ig plasmid expressionvector comprising a nucleotide sequence encoding a soluble extracellulardomain of a human B7-1/IgG1 domain fusion protein. A nucleotide sequenceencoding the extracellular domain of a NCSM polypeptide (or fragmentthereof) can be substituted for the human B7-1-ECD sequence.

FIG. 18 is a photographic representation of an SDS-PAGE gel analysis ofaffinity purified CD28BP-15 ECD-Ig, CTLA-4BP 5X4-12C ECD-Ig, and WThuman B7-1 ECD-Ig fusion proteins. Molecular weight standards are shownon the left.

FIG. 19 is a photograph of a Western blot analysis.

FIGS. 20A and 20E are graphs depicting T cell proliferation responses(measured via ³H thymidine uptake (counts per minute (CPM)) generated byco-culturing various crosslinked (multimeric) soluble NCSM-ECD fusionproteins and hB7-1-ECD fusion proteins with purified T cells in thepresence of soluble anti-CD3 mAbs. FIG. 20B is a graph depicting T cellproliferation responses generated by co-culturing variousnon-crosslinked soluble NCSM-ECD with peripheral blood mononuclear cells(PBMCs). FIGS. 20C and 20D are graphs depicting T cell proliferationresponses induced by co-culturing various non-crosslinked solubleNCSM-ECDs with PBMC and phytohemagglutinin (PHA). FIG. 20F is a graphdepicting proliferation of T cells, measuring ³H thymidine uptake in amixed lymphocyte reaction.

FIG. 21 illustrates an exemplary pMax Vax10.1 plasmid expression vector.

FIG. 22A illustrates an exemplary pMax Vax10.1 plasmid expression vectorthat comprises a nucleotide sequence encoding a CD28BP polypeptide. FIG.22B illustrates a bicistronic pMax Vax10.1 plasmid expression vectorthat comprises a nucleotide sequence encoding a CD28BP polypeptide and anucleotide sequence encoding the cancer antigen EpCam/KSA. Positions ofvarious components of the vectors, including the promoter(s), kanamycinresistant gene, ColE1 replication of origin, BGH poly A adenylationsequences and restriction sites are shown.

FIGS. 23A–23B are histograms depicting the binding of full-lengthhB7-1-Tyr65His variant, CTLA-4BP 5x4-12c (gray histogram), and hB7-1(gray histogram) to either soluble CD28-Ig or CTLA-4-Ig.

FIG. 24 shows a T cell proliferation assay (³H thymidine uptake measuredin counts per minute) using 293 HEK cells transfected with pCDNA or witha nucleic acid sequence encoding full-length hB7-1 (SEQ ID NO:278),hB7-1-Tyr65His polypeptide variant, full-length CTLA-4BP 5x4-12c (SEQ IDNO:39) (clone 12c), or full-length CD28BP-15 (SEQ ID NO:19); 293 cellsalone (with no DNA) were also assayed for their ability to induce humanT-cell proliferation.

FIGS. 25A–25C are graphs depicting T cell proliferation responsesgenerated by co-culturing PBMCs, tetanus toxoid (antigen) and increasingconcentrations of soluble proteins of non-crosslinked CD28BP-15-ECD-Ig,CD28BP-15-ECD, WT huB7.1-ECD-Ig, WT huB7.1-ECD, commercial CTLA-4-Igreceptor/ligand (R&D Systems), and control human IgG antibody.

DETAILED DESCRIPTION OF THE INVENTION

The expression of B7-1 has been shown to be an important mechanism ofimmune responses in mammals, including humans. It is believed that atleast two signals are required for activation of T cells byantigen-bearing target cells: 1) an antigen-specific signal, deliveredthrough the T cell receptor (TCR); and 2) an antigen-independent orco-stimulatory signal that leads to the production of lymphokineproducts (Hodge et al. (1994) Cancer Res. 54:5552–5555). B7-1, which istypically expressed on antigen-presenting cells (APC), has beendetermined to be a ligand for two T cell surface antigen receptors: CD28and CTLA-4. Both receptors are present on T cells, although they areexpressed at different times and in different amounts. T cell activationis a prerequisite for all specific immune responses. However, if onlyone T cell activation signal is received by a T cell, activation willlikely not occur, and anergy may result. For example, many tumor cellsdo not express B7-1. Consequently, even when a tumor cell expresses apotential rejection antigen, it is not likely that it will be able toactivate an antitumor T cell response. Id. For T cell activation andenhanced immune response, an additional antigen-independent signal, suchas from B7-1, is believed necessary.

The human CD28 receptor and human CTLA-4 receptor are naturallyactivated in human cells by B7-1. In some studies, the reported bindingaffinities of CTLA-4 and CD28 to WT hB7-1 were found to be about0.2–0.4×10⁻⁶ M and about 4×10⁻⁶ M, respectively (van der Merwe et al.(1997) J. Exp. Med. 185:393; Ikemizu et al. (2000) Immunity 12:51).However, different studies have reported different binding affinities.

The amino acid sequence of full-length WT hB7-1 comprises 288 aminoacids (GenBank Protein Access. No. P33681) (SEQ ID NO:278). The signalpeptide of WT hB7-1 (which is cleaved in the secreted form) typicallycomprises amino acid residues 1–34, the extracellular domain (ECD)comprises amino acid residues 35–242, the transmembrane domain comprisesamino acid residues 243–263, and the cytoplasmic domain comprises aminoacid residues 264–288. The mature form of hB7-1, which has a total of254 amino acids, comprises amino acid residues 35–288 (the full-lengthsequence without the signal peptide), and begins with the amino acidsequence: valine-isoleucine-histidine-valine. If desired, the aminoacids of the mature form can be numbered beginning with the Val of theVal-Ile-His-Val sequence, designating Val as the first residue (e.g.,the ECD comprises amino acid residues numbered 1–208). In anotheraspect, the ECD of hB7-1 comprises amino acid residues 1–208, thetransmembrane domain comprises amino acid residues 209–235, and thecytoplasmic domain comprises amino acid residues 236–254 of thefull-length mature hB7-1 sequence when numbered beginning with the Valof the Val-Ile-His-Val sequence of the mature sequence as describedabove. See, e.g., U.S. Pat. No. 6,071,716. There are eight possibleglycosylation sites (Asn-X-Ser/Thr) in hB7-1 ECD. The transmembranedomain includes at least 3 cysteine residues that may be involved inbinding to other polypeptides or lipid derivatization. Id.

In yet another aspect, for hB7-1, the ECD comprises amino acid residues35–251, the TMD comprises amino acid residues 252–267, and the CDcomprises amino acid residues 268–288. From the full-length NCSMpolypeptides set forth herein, the subsequences corresponding to thesignal peptide, ECD, TMD, and CD of the NCSM polypeptide, respectively,can be determined as described in greater detail below by, e.g.,alignment of the NCSM amino acid sequence with hB7-1 amino acid sequenceor by using known methods to predict the location and/or cleavage sitesof such sequences (e.g., methods to predict signal peptide cleavagesites, computer program to determine, e.g., regions of hydrophobicitycorresponding to the amino acid residues of a TMD, etc.).According toone study, the nucleic acid sequence of WT hB7-1 comprises 1491 basepairs and is set forth in U.S. Pat. No. 6,071,716 (see SEQ ID NO:1therein). An alignment of the hB7-1 nucleic acid sequence with itscorresponding full-length amino acid sequence is also shown in U.S. Pat.No. 6,071,716 (see SEQ ID NO:1 therein).

Using the nucleotide sequences of human B7-1 and other selectedmammalian B7-1 molecules, we generated recombination nucleotidesencoding recombinant chimeric co-stimulatory peptides molecules havingaltered properties as compared those of WT hB7. This embodiment andothers are described in detail below. FIG. 1 illustrates an interactionbetween an NCSM molecule of the invention, as expressed of an APC cell,and corresponding receptor, expressed on a T cell.

Definitions

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

A “polynucleotide sequence” is a nucleic acid which comprises a polymerof nucleic acid residues or nucleotides (A,C,T,U,G, etc. or naturallyoccurring or artificial nucleotide analogues), or a character stringrepresenting a nucleic acid, depending on context. Either the givennucleic acid or the complementary nucleic acid can be determined fromany specified polynucleotide sequence.

A “polypeptide sequence” is a polymer of amino acids (a protein,polypeptide, etc., comprising amino acid residues) or a character stringrepresenting an amino acid polymer, depending on context. Given thedegeneracy of the genetic code, one or more nucleic acids, or thecomplementary nucleic acids thereof, that encode a specific polypeptidesequence can be determined from the polypeptide sequence.

A nucleic acid, protein, peptide, polypeptide, or other component is“isolated” when it is partially or completely separated from componentswith which it is normally associated (other peptides, polypeptides,proteins (including complexes, e.g., polymerases and ribosomes which mayaccompany a native sequence), nucleic acids, cells, synthetic reagents,cellular contaminants, cellular components, etc.), e.g., such as fromother components with which it is normally associated in the cell fromwhich it was originally derived. A nucleic acid, polypeptide, or othercomponent is isolated when it is partially or completely recovered orseparated from other components of its natural environment such that itis the predominant species present in a composition, mixture, orcollection of components (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In preferredembodiments, the preparation consists of more than about 70% or 75%,typically more than about 80%, or preferably more than about 90% of theisolated species.

In one aspect, a “substantially pure” or “isolated” nucleic acid (e.g.,RNA or DNA), polypeptide, protein, or composition also means where theobject species (e.g., nucleic acid or polypeptide) comprises at leastabout 50, 60, or 70 percent by weight (on a molar basis) of allmacromolecular species present. A substantially pure or isolatedcomposition can also comprise at least about 80, 90, or 95 percent byweight of all macromolecular species present in the composition. Anisolated object species can also be purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of derivatives of a single macromolecular species. The term“purified” generally denotes that a nucleic acid, polypeptide, orprotein gives rise to essentially one band in an electrophoretic gel. Ittypically means that the nucleic acid, polypeptide, or protein is atleast about 50% pure, 60% pure, 70% pure, 75% pure, more preferably atleast about 85% pure, and most preferably at least about 99% pure.

The term “isolated nucleic acid” may refer to a nucleic acid (e.g., DNAor RNA) that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (i.e., one at the 5′and one at the 3′ end) in the naturally occurring genome of the organismfrom which the nucleic acid of the invention is derived. Thus, this termincludes, e.g., a cDNA or a genomic DNA fragment produced by polymerasechain reaction (PCR) or restriction endonuclease treatment, whether suchcDNA or genomic DNA fragment is incorporated into a vector, integratedinto the genome of the same or a different species than the organism,including, e.g., a virus, from which it was originally derived, linkedto an additional coding sequence to form a hybrid gene encoding achimeric polypeptide, or independent of any other DNA sequences. The DNAmay be double-stranded or single-stranded, sense or antisense.

The term “recombinant” when used with reference, e.g., to a cell,nucleotide, vector, protein, or polypeptide typically indicates that thecell, nucleotide, or vector has been modified by the introduction of aheterologous (or foreign) nucleic acid or the alteration of a nativenucleic acid, or that the protein or polypeptide has been modified bythe introduction of a heterologous amino acid, or that the cell isderived from a cell so modified. Recombinant cells express nucleic acidsequences (e.g., genes) that are not found in the native(non-recombinant) form of the cell or express native nucleic acidsequences (e.g., genes) that would be abnormally expressedunder-expressed, or not expressed at all. The term “recombinant” whenused with reference to a cell indicates that the cell replicates aheterologous nucleic acid, or expresses a peptide or protein encoded bya heterologous nucleic acid. Recombinant cells can contain genes thatare not found within the native (non-recombinant) form of the cell.Recombinant cells can also contain genes found in the native form of thecell wherein the genes are modified and re-introduced into the cell byartificial means. The term also encompasses cells that contain a nucleicacid endogenous to the cell that has been modified without removing thenucleic acid from the cell; such modifications include those obtained bygene replacement, site-specific mutation, and related techniques.

A “recombinant polynucleotide” or a “recombinant polypeptide” is anon-naturally occurring polynucleotide or polypeptide that includesnucleic acid or amino acid sequences, respectively, from more than onesource nucleic acid or polypeptide, which source nucleic acid orpolypeptide can be a naturally occurring nucleic acid or polypeptide, orcan itself have been subjected to mutagenesis or other type ofmodification. A nucleic acid or polypeptide may be deemed “recombinant”when it is artificial or engineered, or derived from an artificial orengineered polypeptide or nucleic acid. A recombinant nucleic acid(e.g., DNA or RNA) can be made by the combination (e.g., artificialcombination) of at least two segments of sequence that are not typicallyincluded together, not typically associated with one another, or areotherwise typically separated from one another. A recombinant nucleicacid can comprise a nucleic acid molecule formed by the joining togetheror combination of nucleic acid segments from different sources and/orartificially synthesized. A “recombinant polypeptide” (or “recombinantprotein”) often refers to a polypeptide (or protein) that results from acloned or recombinant nucleic acid or gene. The source polynucleotidesor polypeptides from which the different nucleic acid or amino acidsequences are derived are sometimes homologous (i.e., have, or encode apolypeptide that encodes, the same or a similar structure and/orfunction), and are often from different isolates, serotypes, strains,species, of organism or from different disease states, for example.

The term “recombinantly produced” refers to an artificial combinationusually accomplished by either chemical synthesis means, recursivesequence recombination of nucleic acid segments or other diversitygeneration methods (such as, e.g., shuffling) of nucleotides, ormanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques known to those of ordinary skill in the art.“Recombinantly expressed” typically refers to techniques for theproduction of a recombinant nucleic acid in vitro and transfer of therecombinant nucleic acid into cells in vivo, in vitro, or ex vivo whereit may be expressed or propagated.

A “recombinant expression cassette” or simply a n “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

By “immune response” is intended an alteration of an organism's orsubject's immune system in response to an immunomodulatory agent,immunogen, or antigen that may include, but is not limited to, antibodyproduction, induction of cell-mediated immunity, complement activation,development of immunological tolerance, inhibition of an immuneresponse, or breaking of immunological tolerance. An “immunomodulatoryagent” or “immunomodulatory molecule” modulates an immune response. An“immunogen” refers generally to a substance capable of provoking oraltering an immune response, and includes, but is not limited to, e.g.,immunogenic proteins, polypeptides, and peptides; antigens and antigenicpeptide fragments thereof; and nucleic acids having immunogenicproperties or encoding, e.g., polypeptides having such properties.

An “immunogen” refers to a substance capable of provoking an immuneresponse, and includes, e.g., antigens, autoantigens that play a role ininduction of autoimmune diseases, and tumor-associated antigensexpressed on cancer cells. An immune response generally refers to thedevelopment of a cellular or antibody-mediated response to an agent,such as an antigen or fragment thereof or nucleic acid encoding suchagent. In some instances, such a response comprises a production of atleast one or a combination of CTLs, B cells, or various classes of Tcells that are directed specifically to antigen-presenting cellsexpressing the antigen of interest.

By “modulation” or “modulating” an immune response of a subject isintended that the immune response of the subject is altered. Forexample, “modulation” or “modulating” an immune response of a subjectmeans, e.g., that the immune response is stimulated, invoked, decreased,increased, enhanced, or otherwise altered. The immunological responsemay be skewed or shifted from Th1 to Th2 or vice versa to optimizeprotection and reduce unwanted side effects of the immunologicalresponse. An immunomodulatory agent or molecule modulates an immuneresponse. An immunomodulatory agent has an immunomodulatory activity. Amodulated immune response of a subject to whom an immunomodulatory agentis administered differs from the immune response of an untreated subjectto whom the immunomodulatory has not been administered by 0.1%, 0.5%,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.Modulation of an immune response in a subject can be assessed by meansknown to those skilled in the art, including those described below.

“Tolerance” refers to a state of diminished or lack of immunologicalresponsiveness. Tolerance typically defines an absent or diminished orlessened capacity of a subject to mount an immune response against agiven antigen, usually the result of, e.g., contact between the subjectand a target antigen under non-immunizing conditions.

“Anergy” refers to a state of diminished reactivity to one or moreantigens. For example, anergy state is often characterized by diminishedT cell responses, e.g., proliferation or IL-2 production, when specificT cells are restimulated under otherwise stimulatory conditions.

An “antigen” refers to a substance that is capable of inducing an immuneresponse (e.g., humoral and/or cell-mediated) in a host, including, butnot limited to, eliciting the formation of antibodies in a host, orgenerating a specific population of lymphocytes reactive with thatsubstance. Antigens are typically macromolecules (e.g., proteins andpolysaccharides) that are foreign to the host.

A “subsequence” or “fragment” is any portion of an entire sequence, upto and including the complete sequence. Thus, a “subsequence” refers toa sequence of nucleic acids or amino acids that comprises a part of alonger sequence of nucleic acids (e.g., polynucleotide) or amino acids(e.g., polypeptide) respectively.

An “adjuvant” refers to a substance that enhances an immune response,including, for example, but not limited to, an antigen'simmune-stimulating properties or the pharmacological effect(s) of acompound or drug. An adjuvant may non-specifically enhance an immuneresponse, e.g., the immune response to an antigen. “Freund's CompleteAdjuvant,” for example, is an emulsion of oil and water containing animmunogen, an emulsifying agent and mycobacteria. Another example,“Freund's incomplete adjuvant,” is the same, but without mycobacteria.An adjuvant may comprise oils, emulsifiers, killed bacteria, aluminumhydroxide, or calcium phosphate (e.g., in gel form), or combinationsthereof. An adjuvant may be administered into a subject (e.g., viainjection intramuscularly or subcutaneously) in an amount sufficient toproduce antibodies.

Numbering of a given amino acid polymer or nucleotide polymer“corresponds to numbering” of a selected amino acid polymer or nucleicacid polymer when the position of any given polymer component (e.g.,amino acid residue, nucleotide residue) is designated by reference tothe same or an equivalent residue position in the selected amino acid ornucleotide polymer, rather than by the actual position of the componentin the given polymer. Thus, for example, the numbering of a given aminoacid position in a given polypeptide sequence corresponds to the same orequivalent amino acid position in a selected polypeptide sequence usedas a reference sequence.

A vector is a component or composition for facilitating celltransduction or transfection by a selected nucleic acid, or expressionof the nucleic acid in the cell. Vectors include, e.g., plasmids,cosmids, viruses, YACs, bacteria, poly-lysine, etc. An “expressionvector” is a nucleic acid construct or sequence, generated recombinantlyor synthetically, with a series of specific nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. The expression vector typically includes a nucleic acid to betranscribed operably linked to a promoter. The nucleic acid to betranscribed is typically under the direction or control of the promoter.

“Substantially the entire length of a polynucleotide sequence” or“substantially the entire length of a polypeptide sequence” refers to atleast about 50%, generally at least about 60%, 70%, or 75%, usually atleast about 80%, or typically at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or more of a length of a polynucleotidesequence or polypeptide sequence.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature as distinct from being artificiallyproduced by man. A polypeptide or polynucleotide sequence that ispresent in an organism (including viruses, bacteria, protozoa, insects,plants or mammalian tissue) that can be isolated from a source in natureand which has not been intentionally modified by man in the laboratoryis naturally occurring. Non-naturally occurring as applied to an objectmeans that the object is not naturally-occurring—i.e., the object cannotbe found in nature as distinct from being artificially produced by man.

The term “immunoassay” includes an assay that uses an antibody orimmunogen to bind or specifically bind an antigen. The immunoassay istypically characterized by the use of specific binding properties of aparticular antibody to isolate, target, and/or quantify the antigen.

The term “homology” generally refers to the degree of similarity betweentwo or more structures. The term “homologous sequences” refers toregions in macromolecules that have a similar order of monomers. Whenused in relation to nucleic acid sequences, the term “homology” refersto the degree of similarity between two or more nucleic acid sequences(e.g., genes) or fragments thereof. Typically, the degree of similaritybetween two or more nucleic acid sequences refers to the degree ofsimilarity of the composition, order, or arrangement of two or morenucleotide bases (or other genotypic feature) of the two or more nucleicacid sequences. The term “homologous nucleic acids” generally refers tonucleic acids comprising nucleotide sequences having a degree ofsimilarity in nucleotide base composition, arrangement, or order. Thetwo or more nucleic acids may be of the same or different species orgroup. The term “percent homology” when used in relation to nucleic acidsequences, refers generally to a percent degree of similarity betweenthe nucleotide sequences of two or more nucleic acids.

When used in relation to polypeptide (or protein) sequences, the term“homology” refers to the degree of similarity between two or morepolypeptide (or protein) sequences (e.g., genes) or fragments thereof.Typically, the degree of similarity between two or more polypeptide (orprotein) sequences refers to the degree of similarity of thecomposition, order, or arrangement of two or more amino acid of the twoor more polypeptides (or proteins). The two or more polypeptides (orproteins) may be of the same or different species or group. The term“percent homology” when used in relation to polypeptide (or protein)sequences, refers generally to a percent degree of similarity betweenthe amino acid sequences of two or more polypeptide (or protein)sequences. The term “homologous polypeptides” or “homologous proteins”generally refers to polypeptides or proteins, respectively, that haveamino acid sequences and functions that are similar. Such homologouspolypeptides or proteins may be related by having amino acid sequencesand functions that are similar, but are derived or evolved fromdifferent or the same species using the techniques described herein.

The term “subject” as used herein includes, but is not limited to, anorganism; a mammal, including, e.g., a human, non-human primate (e.g.,baboon, orangutan, monkey), mouse, pig, cow, goat, cat, rabbit, rat,guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; anon-mammal, including, e.g., a non-mammalian vertebrate, such as a bird(e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.

The term “pharmaceutical composition” means a composition suitable forpharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount of anactive agent and a carrier, including, e.g., a pharmaceuticallyacceptable carrier.

The term “effective amount” means a dosage or amount sufficient toproduce a desired result. The desired result may comprise an objectiveor subjective improvement in the recipient of the dosage or amount.

A “prophylactic treatment” is a treatment administered to a subject whodoes not display signs or symptoms of a disease, pathology, or medicaldisorder, or displays only early signs or symptoms of a disease,pathology, or disorder, such that treatment is administered for thepurpose of diminishing, preventing, or decreasing the risk of developingthe disease, pathology, or medical disorder. A prophylactic treatmentfunctions as a preventative treatment against a disease or disorder. A“prophylactic activity” is an activity of an agent, such as a nucleicacid, vector, gene, polypeptide, protein, substance, or compositionthereof that, when administered to a subject who does not display signsor symptoms of pathology, disease or disorder, or who displays onlyearly signs or symptoms of pathology, disease, or disorder, diminishes,prevents, or decreases the risk of the subject developing a pathology,disease, or disorder. A “prophylactically useful” agent or compound(e.g., nucleic acid or polypeptide) refers to an agent or compound thatis useful in diminishing, preventing, treating, or decreasingdevelopment of pathology, disease or disorder.

A “therapeutic treatment” is a treatment administered to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms of pathology, disease, ordisorder. A “therapeutic activity” is an activity of an agent, such as anucleic acid, vector, gene, polypeptide, protein, substance, orcomposition thereof, that eliminates or diminishes signs or symptoms ofpathology, disease or disorder, when administered to a subject sufferingfrom such signs or symptoms. A “therapeutically useful” agent orcompound (e.g., nucleic acid or polypeptide) indicates that an agent orcompound is useful in diminishing, treating, or eliminating such signsor symptoms of a pathology, disease or disorder.

The term “gene” broadly refers to any segment of DNA associated with abiological function. Genes include coding sequences and/or regulatorysequences required for their expression. Genes also includenon-expressed DNA nucleic acid segments that, e.g., form recognitionsequences for other proteins (e.g., promoter, enhancer, or otherregulatory regions). Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, molecular biology, nucleic acidchemistry, and protein chemistry described below are those well knownand commonly employed by those of ordinary skill in the art. Standardtechniques, such as described in Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1–3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994, supplemented through 1999)(hereinafter “Ausubel”), are used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture methods, and transgeneincorporation, e.g., electroporation, injection, gene gun, impressingthrough the skin, and lipofection. Generally, oligonucleotide synthesisand purification steps are performed according to specifications. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references which areprovided throughout this document. The procedures therein are believedto be well known to those of ordinary skill in the art and are providedfor the convenience of the reader.

As used herein, an “antibody” refers to a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The term antibody is used to meanwhole antibodies and binding fragments thereof. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 KDa) and one “heavy” chain (about 50–70 KDa). The N-terminusof each chain defines a variable region of about 100 to 110 or moreamino acids primarily responsible for antigen recognition. The termsvariable light chain (VL) and variable heavy chain (VH) refer to theselight and heavy chains, respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′2, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)2 dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region. The Fc portion of the antibody molecule correspondslargely to the constant region of the immunoglobulin heavy chain, and isresponsible for the antibody's effector function (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, NY (1993), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments may be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein also includes antibody fragmentseither produced by the modification of whole antibodies or synthesizedde novo using recombinant DNA methodologies.

Antibodies also include single-armed composite monoclonal antibodies,single chain antibodies, including single chain Fv (sFv) antibodies inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide,as well as diabodies, tribodies, and tetrabodies (Pack et al. (1995) JMol Biol 246:28; Biotechnol 11:1271; and Biochemistry 31:1579). Theantibodies are, e.g., polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments, fragments produced by an Fab expressionlibrary, or the like.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An “antigen-binding fragment” of an antibody is a peptide or polypeptidefragment of the antibody that binds an antigen. An antigen-binding siteis formed by those amino acids of the antibody that contribute to, areinvolved in, or affect the binding of the antigen. See Scott, T. A. andMercer, E. I., Concise Encyclopedia: Biochemistry and Molecular Biology(de Gruyter, 3d ed. 1997), and Watson, J. D. et al., Recombinant DNA (2ded. 1992) [hereinafter “Watson, Recombinant DNA”], each of which isincorporated herein by reference in its entirety for all purposes.

The term “screening” describes, in general, a process that identifiesoptimal molecules of the present invention, such as, e.g., the NCSMpolypeptide and proteins, fragments and homologues thereof, and relatedfusion polypeptides and proteins including the same, nucleic acidsencoding all such molecules. Several properties of these respectivemolecules can be used in selection and screening, for example, anability of a respective molecule to bind to a receptor, to alter animmune response, e.g., induce or inhibit a desired immune response, in atest system or an in vitro, ex vivo or in vivo application (e.g., induceor inhibit a T cell proliferation response in conjunction withcostimulation of T cell receptor/CD3 (by, e.g., an antigen or anti-CD3antibody)), or to bind a first receptor with equal, greater, or lessbinding affinity relative to a second receptor compared to the bindingaffinity of a control molecule (e.g., a wild-type B7-1 or co-stimulatorymolecule) for the first and second receptors, as measured by therespective molecule's first receptor/second receptor binding affinityratio (or its reciprocal), compared to the control molecule's firstreceptor/second receptor binding affinity ratio. In the case ofantigens, several properties of the antigen can be used in selection andscreening including antigen expression, folding, stability,immunogenicity and presence of epitopes from several related antigens.Selection is a form of screening in which identification and physicalseparation are achieved simultaneously by expression of a selectionmarker, which, in some genetic circumstances, allows cells expressingthe marker to survive while other cells die (or vice versa). Screeningmarkers include, for example, luciferase, beta-galactosidase and greenfluorescent protein, and the like. Selection markers include drug andtoxin resistance genes, and the like. Because of limitations in studyingprimary immune responses in vitro, in vivo or ex vivo studies areparticularly useful screening methods. In these studies, geneticvaccines or expression vectors that include sequences encoding one ormore respective NCSM polypeptides, are first introduced to test animals,and the immune responses are subsequently studied by analyzingprotective immune responses or by studying the quality or strength ofthe induced immune response using lymphoid cells derived from theimmunized animal. Alternatively, the NCSM polypeptide itself or asoluble form thereof (e.g., the ECD of the polypeptide or a fragmentthereof alone or in a fusion protein) is introduced to the test animal.Although spontaneous selection can and does occur in the course ofnatural evolution, in the present methods selection is performed by man.

A “specific binding affinity” between two molecules, e.g., a ligand anda receptor, means a preferential binding of one molecule for another ina mixture of molecules. The binding of the molecules is typicallyconsidered specific if the binding affinity is about 1×10² M⁻¹ to about1×10⁷ M⁻¹ (i.e., about 10⁻²–10⁻⁷ M) or greater.

A “binding affinity ratio” refers to a relative ratio of the bindingaffinity of a molecule of interest (e.g., a recombinant ligand, such asa NSCM polypeptide) for a first molecule (e.g., a first receptor, suchas CD28 receptor) to the binding affinity of the same molecule ofinterest to a second molecule (e.g., a second receptor, such as CTLA-4receptor). In one aspect, the relative binding affinity ratio may bedetermined by visual inspection, such as by, eg., examining a FACSbinding profile that displays the binding affinity profile of themolecule of interest to both receptors, and evaluating the degree ofrelative binding of the molecule of interest to each of the first andsecond receptors. The results of this determination can be compared witha similar examination and evaluation of a FACS binding affinity profiledisplaying the binding affinity of a control molecule (e.g., wild-typeligand, such as a WT human, primate, or mammalian B7-1) to bothreceptors, wherein the degree of relative binding of the controlmolecule to each of the receptors is evaluated. These and otherprocedures described below can be used to determine a CD28/CTLA-4binding affinity ratio for a CD28BP polypeptide of the present inventionand a CTLA-4/CD28 binding affinity ratio for a CTLA-4BP polypeptide ofthe present invention. Alternatively, a binding affinity ratio can bedetermined by making a ratio between a quantitative measurement of thebinding affinity of the molecule of interest (e.g., ligand) for thefirst receptor and a quantitative measurement of the binding affinity ofthe molecule of interest for the second receptor using known proceduresfor measuring binding affinities. For example, known methods formeasuring the binding affinity of human (or other mammalian) B7-1 foreach of CD28 and CTLA-4 receptors can be used.

An “exogenous” nucleic acid,” “exogenous DNA segment,” “heterologoussequence,” or “heterologous nucleic acid,” as used herein, is one thatoriginates from a source foreign to the particular host cell, or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell, but has been modified. Modification of aheterologous sequence in the applications described herein typicallyoccurs through the use of recursive sequence recombination. The termsrefer to a DNA segment which is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605–2608;Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91–98).The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

“Nucleic acid derived from a gene” refers to a nucleic acid for whosesynthesis the gene, or a subsequence thereof, has ultimately served as atemplate. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc., are all derived from the geneand detection of such derived products is indicative of the presenceand/or abundance of the original gene and/or gene transcript in asample.

A nucleic acid is “operably linked” with another nucleic acid sequencewhen it is placed into a functional relationship with another nucleicacid sequence. For instance, a promoter or enhancer is operably linkedto a coding sequence if it increases the transcription of the codingsequence. Operably linked means that the DNA sequences being linked aretypically contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. However, since enhancersgenerally function when separated from the promoter by several kilobasesand intronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

The term “cytokine” includes, for example, interleukins, interferons,chemokines, hematopoietic growth factors, tumor necrosis factors andtransforming growth factors. In general these are small molecular weightproteins that regulate maturation, activation, proliferation, anddifferentiation of cells of the immune system.

A “variant” of a polypeptide is a polypeptide that differs in one ormore amino acid residues from a parent or reference polypeptide, usuallyin at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15amino acid or more residues.

A “variant” of a nucleic acid is a nucleic acid that differs in one ormore nucleic acid residues from a parent or reference nucleic acid,usually in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 17, 20, 21, 24, 27, 30, 33, 36, 39, 40, 45, 50 or more nucleic acidresidues.

Various additional terms are defined or otherwise characterized herein.

Polynucleotides of the Invention

NCSM Polynucleotide Sequences

The invention provides isolated or recombinant NCSM polypeptides andfragments thereof, and isolated or recombinant polynucleotides encodingsaid polypeptides and fragments thereof. The term “NCSM polynucleotide”is intended throughout to include nucleic acid fragments, homologues,and variants of the polynucleotide sequences specifically disclosedherein unless otherwise noted.

In one aspect, the polynucleotides and polypeptides of the inventionwere made in two rounds of recursive sequence recombination using DNArecombination methods and formats described below. In preparation, priorto the rounds, cDNAs encoding, e.g., primate (rhesus monkey, baboon, andorangutan), cow, cat, and rabbit, B7-1 related sequences were clonedfrom their respective species, either from cell lines or peripheralblood. The cDNAs of the invention encoding baboon B7-1 and orangutanB7-1 are examples of previously unknown WT B7-1 polynucleotides. Baboonand orangutan B7-1 have CD28 and CTLA-4 binding properties and T cellproliferation properties similar to those of hB7-1 (data not shown). Thepolynucleotide sequences encoding baboon (SEQ ID NO:46) and orangutan(SEQ ID NO:47) B7-1, corresponding baboon B7-1 (SEQ ID NO:93) andorangutan (SEQ ID NO:94) B7-1 polypeptides, and homologues, fragments(e.g., ECD), fusion proteins thereof, are aspects of the invention.

In Round 1, the cDNAs encoding human, primate, cow, cat, and rabbit B7-1were recursively recombined to form libraries comprising two or morerecombinant polynucleotides. Other methods for obtaining libraries ofrecombinant polynucleotides (including NCSM polynucleotides) and/or forobtaining diversity in nucleic acids used as the substrates forrecursive sequence recombination are also described infra. The librariesof Round 1 were initially screened via three methods. An initialscreening sorted the pooled recombined clones based on preferentialbinding ability to soluble CD28 and CTLA-4 receptor fusion proteins. Asecond screening selected individual clones based on the ability to bindto either CD28 or CTLA-4. A third screening tested the individual clonesfrom the second screen based on the ability to induce or inhibit T cellproliferation in conjunction with costimulation of T cell receptor/CD3(by, e.g., an antigen or anti-CD3 Ab). Exemplary NCSM nucleic acids fromRound 1 encoding NCSM polypeptides having a preferential or similarbinding to CD28 relative to CTLA-4, designated as CD28 binding proteins(“CD28BP”), as compared to the binding of WT hB7-1 to CD28 relative toCTLA-4, and/or having an ability to induce proliferation of T cells withT cell receptor co-engagement (e.g., in conjunction with stimulation ofT cell receptor by, e.g., an antigen or anti-CD3 Ab) are shown in SEQ IDNOS:1–4, which encode NCSM polypeptides identified herein as SEQ IDNOS:48–51. Exemplary NCSM nucleic acids from Round 1 encoding NCSMpolypeptides having a preferential or similar binding to CTLA-4 relativeto CD28, designated as CTLA-4 binding proteins (“CTLA-4BP”), as comparedto the binding of WT hB7-1 to CTLA-4 relative to CD28, and/or having anability to inhibit proliferation of T cells with T cell receptorco-engagement (e.g., in conjunction with stimulation of T cell receptorby, e.g., an antigen or anti-CD3 mAb) are shown in SEQ ID NOS:22–26,which encode NCSM polypeptides identified herein as SEQ ID NOS:69–73.

Exemplary clones from Round 1 were further recombined in Round 2 to formrecombinant polynucleotide libraries. Similar screenings were done as inRound 1 for the polynucleotide clones produced in Round 2. Exemplaryrecursively recombined NCSM nucleic acids encoding NCSM polypeptideshaving a preferential or similar binding to CD28 relative to CTLA-4 ascompared to the binding of WT hB7-1 to CD28 relative to CTLA-4 (e.g.,CD28BP polypeptides), and/or having an ability to induce proliferationof T cells in conjunction with stimulation of T cell receptor in SEQ IDNOS:5–21 and SEQ ID NOS:95–142, which encode NCSM polypeptidesidentified herein as SEQ ID NOS:52–68, SEQ ID NOS:174–221. Additionalidentified recombinant CD28BP polypeptides that were identified includeSEQ ID NOS:283–285 and 289–293.

Exemplary nucleic acids from Round 2 encoding NCSM polypeptides havingpreferential binding to CTLA-4 relative to CD28 as compared to thebinding of WT hB7-1 to CTLA-4 relative to CD28 (e.g., CTLA-4BPpolypeptides), and/or having an ability to inhibit proliferation of Tcells in conjunction with stimulation of T cell receptor are describedin SEQ ID NOS:27–45 and SEQ ID NOS:143–262, which encode NCSMpolypeptides identified herein as SEQ ID NOS:74–92 and SEQ IDNOS:222–252, and 263–272. Additional recombinant CTLA-4BP polypeptidesthat were identified include SEQ ID NOS: 286–288.

The term “preferential binding” in reference to an ability of an NCSMpolypeptide of the invention to bind or specifically bind a CD28receptor and/or CTLA-4 receptor typically refers to a preferentialability of the NCSM polypeptide to bind one or both of these tworeceptors (or to bind one such receptor relative to the other) ascompared to the ability of a WT B7-1 (e.g., human, primate, or mammalianB7-1) to bind one or both of these two receptors (or to bind one suchreceptor relative to the other). A ligand's preferential binding to areceptor typically refers to a greater, enhanced, or improved binding ofthe ligand to the receptor as compared to the binding of a controlmolecule to the receptor.

The ratio of the relative binding affinity of each CD28BP polypeptidefor CD28 and CTLA-4 was determined and defined as the CD28/CTLA-4binding affinity ratio. A ratio of the relative binding affinity of WThB7-1 for CD28 and CTLA-4 was also determined for comparison. TheCD28/CTLA-4 binding affinity ratios of the CD28BP polypeptides were eachfound to be at least about equal to or greater than the CD28/CTLA-4binding affinity ratio of WT hB7-1. The ratio of the relative bindingaffinity of each CTLA-4BP polypeptide for CD28 and CTLA-4 was alsodetermined (i.e., CTLA-4/CD28 binding affinity ratio). A ratio of therelative binding affinity of WT hB7-1 for each of CTLA-4 and CD28 wasalso determined. The CTLA-4/CD28 binding affinity ratios of the CTLA-4BPpolypeptides were each found to be at least about equal to or greaterthan the CTLA-4/CD28 binding affinity ratio of WT hB7-1.

Assays for detecting the production of specific cytokines were alsoperformed, as described in detail below, to identify NCSMpolynucleotides of the invention encoding NCSM polypeptides of theinvention.

The cloned WT cow B7-1 (SEQ ID NO:280) and WT rabbit B7-1 (SEQ IDNO:281) polypeptides also worked in the binding and T cell functionalassays described herein. Both cow and rabbit B7-1 polypeptides induced aT cell response in human T cells. The relative CD28/CTLA-4 bindingaffinity ratio of WT cow B7-1 polypeptide was found to be significantlygreater than that of WT hB7-1. The relative CD28/CTLA-4 binding affinityratio of WT rabbit B7-1 polypeptide was found to be greater than that ofWT hB7-1 (data not shown).

While NCSM polynucleotides of the present invention were identifiedprincipally using the cell-based proliferation assays, receptor bindingassays, and cytokine production assays described supra and infra, otherassays that rely on alternative means of detection are equally suitable.For example, induction of other visual markers by receptor binding canbe favorably employed. Similarly, direct binding to a receptor, e.g., byBiacore plasmon resonance, can be utilized. Other specific cytokineassays well known in the art and used for analysis of B7-1 molecules canalso be utilized.

Soluble NCSM peptide constructs, including, e.g., extracellular domainsof NCSM polypeptides, or fragments or subsequences thereof, alone orfused to immunoglobulin (Ig) polypeptide sequences, were also made andanalyzed for receptor binding, ability to enhance or reduce an immuneresponse, e.g., inhibit or augment T cell proliferation and/oractivation, and ability to produce specific cytokine or alter or augmenttheir levels. Nucleotide coding sequences for these soluble NCSMpeptides constructs were determined. As described in detail below, suchsoluble NCSM polypeptides are useful in a variety of therapeutic,prophylactic, and/or diagnostic applications and methods.

The NCSM polynucleotides of the present invention that encode NCSMpolypeptides are useful in a variety of applications discussed ingreater detail below. For example, NCSM polynucleotides can beincorporated into expression vectors useful for gene therapy, DNAvaccination, and immunotherapy. Such vectors comprising NCSMpolynucleotides encoding NCSM polypeptides are useful in clinical andmedical applications in which it is desirable to provide specificproliferation/activation or anti-proliferation/inactivation of T cellsthat have encountered their specific antigen. Such vectors comprisingNCSM polynucleotides of the invention are also useful in applicationsdesigned to break or avoid tolerance (e.g., vaccine or T cell adjuvants,treatment of malignant diseases and treatment of chronic infectiousdiseases), as where an enhanced immune response is desirable, orapplications designed to induce tolerance (e.g., autoimmunity, severeallergy/asthma and organ transplantation), as where a decreased immuneresponse is desirable.

CD28BP Polynucleotides

The invention provides nucleic acids that encode NCSM polypeptides andfragments thereof, wherein such polypeptides and fragments thereof bindeither or both of CD28 or CTLA-4 receptor and/or modulates a T cellresponse. The binding of molecules can generally be considered specificif the binding affinity is about 1×10² M⁻¹ to about 1×10⁷ M⁻¹ (i.e.,about 10⁻²- about 10⁻⁷ M) or greater. A “CD28 binding protein” or “CD28binding polypeptide” (“CD28BP”) refers generally to a protein orpolypeptide, or fragment or subsequence thereof (such as, e.g., an ECDor trunECD), that binds to or associates with a CD28 receptor. A “CTLA-4binding protein” or “CTLA-4 binding polypeptide” (“CTLA-4BP”) refersgenerally to a protein or polypeptide, or fragment or subsequencethereof (such as, e.g., an ECD or trunECD), that binds to or associateswith a CTLA-4 receptor. A CD28BP polynucleotide is a nucleic acidsequence that encodes a CD28BP amino acid sequence. A CTLA-4BPpolynucleotide is a nucleic acid sequence that encodes a CTLA-4BP aminoacid sequence. Some such CD28BP polypeptides (or nucleic acid encodingthem) exhibit an ability to induce a T cell proliferation or activationresponse about equal to or greater than that of hB7-1. Some suchCTLA-4BPs (or nucleic acids encoding them) exhibit an ability to inducea T cell proliferation or activation response about equal to or lessthan that of hB7-1.

In one aspect, the invention provides isolated or recombinant nucleicacids that each comprise a polynucleotide sequence selected from: (a) apolynucleotide sequence selected from SEQ ID NOS:1–21 and 95–142, or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence encoding a polypeptide selected from SEQ ID NOS:48–68, 174–221,283–285, and 290–293, or a complementary polynucleotide sequencethereof; (c) a polynucleotide sequence which, but for codon degeneracy,hybridizes under at least stringent or highly stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b);and (d) a polynucleotide sequence comprising all or a fragment of (a),(b), or (c), wherein the fragment encodes a polypeptide having aCD28/CTLA-4 binding affinity ratio about equal to or greater than theCD28/CTLA-4 binding affinity ratio of human B7-1 and/or an ability toinduce a T cell proliferation or activation response about equal to orgreater than that of hB7-1.

Also provided are isolated or recombinant nucleic acids, each comprisinga nucleotide sequence selected from the group of: (a) a nucleotidesequence that encodes an extracellular domain (ECD), said nucleotidesequence comprising an ECD coding subsequence of a polynucleotidesequence selected from the group of SEQ ID NOS:1–21 and 95–142, or acomplementary nucleotide sequence thereof; (b) a nucleotide sequenceencoding an ECD, said ECD comprising an amino acid subsequence of apolypeptide sequence selected from the group of SEQ ID NOS:48–68,174–221, 283–285, and 290–293, or a complementary nucleotide sequencethereof; and (c) a nucleotide sequence that, but for the degeneracy ofthe genetic code, hybridizes under at least stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b),wherein said nucleotide sequence encodes a polypeptide that has aCD28/CTLA-4 binding affinity ratio about equal to or greater than thatof hB7-1 and/or an ability to induce a T cell proliferation responseequal to or greater than that induced by human B7-1. Typically, thenucleotide sequence of (c) hybridizes under at least stringentconditions over substantially the entire length of polynucleotidesequence (a) and encodes a polypeptide that has a CD28/CTLA-4 bindingaffinity ratio greater than that of hB7-1.

Some such nucleic acids further comprise at least a second nucleotidesequence that encodes a signal peptide, said second nucleotide sequenceselected from the group of: (a) a nucleotide sequence comprising asignal peptide coding subsequence of a polynucleotide sequence selectedfrom the group of SEQ ID NOS:1–21 and 95–142, or a complementarynucleotide sequence thereof; (b) a nucleotide sequence encoding a signalpeptide, which comprises an amino acid subsequence of a polypeptidesequence selected from the group of SEQ ID NOS:48–68, 174–221, 283–285,and 290–293, or a complementary nucleotide sequence thereof; (c) anucleotide sequence that, but for the degeneracy of the genetic code,hybridizes under at least stringent conditions over substantially theentire length of polynucleotide sequence (a) or (b), wherein saidnucleotide sequence encodes a polypeptide that has a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than that of hB7-1; and (d) anucleotide sequence encoding a signal peptide of a B7-1 polypeptide. Insome aspects, the nucleotide sequence of (c) hybridizes under at leaststringent conditions over substantially the entire length ofpolynucleotide sequence (a) and encodes a polypeptide that has aCD28/CTLA-4 binding affinity ratio greater than that of hB7-1 and/or anability to induce a T cell proliferation response about equal to orgreater than that induced by human B7-1.

Some such nucleic acids further comprising at least a third nucleotidesequence encoding a transmembrane domain selected from the group of: (a)a nucleotide sequence comprising a TMD coding subsequence of apolynucleotide sequence selected from the group of SEQ ID NOS:1–21 and95–142, or a complementary nucleotide sequence thereof; (b) a nucleotidesequence encoding a TMD, which comprises an amino acid subsequence of apolypeptide sequence selected from the group of SEQ ID NOS:48–68,174–221, 283–285, and 290–293, or a complementary nucleotide sequencethereof; (c) a nucleotide sequence that, but for the degeneracy of thegenetic code, hybridizes under at least stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b),wherein said nucleotide sequence encodes a polypeptide that has aCD28/CTLA-4 binding affinity ratio about equal to or greater than thatof hB7-1; and (d) a nucleotide sequence that encodes a TMD of a B7-1polypeptide. Some polypeptides further comprise at least a fourthnucleotide sequence encoding a cytoplasmic domain selected from thegroup of: (a) a nucleotide sequence comprising a CD coding subsequenceof a polynucleotide sequence selected from the group of SEQ ID NOS:1–21and 95–142, or a complementary nucleotide sequence thereof; (b) anucleotide sequence encoding a CD, which CD comprises an amino acidsubsequence of a polypeptide sequence selected from the group of SEQ IDNOS:48–68, 174–221, 283–285, and 290–293, or a complementary nucleotidesequence thereof; (c) a nucleotide sequence that, but for the degeneracyof the genetic code, hybridizes under at least stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b),wherein said nucleotide sequence encodes a polypeptide that has aCD28/CTLA-4 binding affinity ratio about equal to or greater than thatof hB7-1; and (d) a nucleotide sequence that encodes a CD of a B7-1polypeptide.

In another aspect, the invention provides isolated or recombinantnucleic acids that each comprises a polynucleotide sequence encoding apolypeptide, wherein the encoded polypeptide comprises an amino acidsequence which is (a) substantially identical over at least about 100contiguous amino acid residues of any one of SEQ ID NOS:48–68, 174–221,283–285, and 290–293 and (b) is a non naturally-occurring sequence. Insome instances, the encoded polypeptide is substantially identical overat least about 150 or about 200 contiguous amino acid residues of anyone of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293.

In yet another aspect, the invention provides isolated or recombinantnucleic acids that each comprise a nucleotide sequence coding for apolypeptide comprising the amino acid sequence set forth in any of SEQID NOS:48–68, 174–221, 283–285, and 290–293, or a subsequence thereof,wherein the subsequence comprises at least one of the signal sequence,ECD, transmembrane domain, and cytoplasmic domain of the polypeptide,and wherein the amino acid sequence or subsequence is a nonnaturally-occurring sequence.

For some such isolated or recombinant polypeptides, the polypeptidecomprises an ECD amino acid sequence encoded by an ECD coding nucleotidesequence, and the ECD coding nucleotide sequence comprises a nucleotidesequence that, but for codon degeneracy, hybridizes under at leaststringent conditions over substantially the entire length of the ECDcoding nucleotide sequence of a polynucleotide sequence selected fromany of SEQ ID NOS:1–21 and 95–142 or the nucleotide coding sequence thatencodes the ECD of a polypeptide selected from any of SEQ ID NOS:48–68,174–221, 283–285, and 290–293. Some such isolated or recombinantpolypeptides further comprises a signal peptide amino acid sequenceencoded by a signal peptide coding nucleotide sequence. The signalpeptide coding nucleotide sequence selected from the group of: (a) anucleotide sequence of a polynucleotide sequence selected from any ofSEQ ID NOS:1–21 and 95–142, wherein said nucleotide sequence encodes asignal peptide; (b) a nucleotide sequence that encodes the signalpeptide of a polypeptide selected from any of SEQ ID NOS:48–68, 174–221,283–285, and 290–293; and (c) a nucleotide sequence which, but for codondegeneracy, hybridizes under at least stringent conditions oversubstantially the entire length of a nucleotide sequence (a) or (b).

Some such isolated or recombinant polypeptides further comprise atransmembrane domain (TMD) amino acid sequence encoded by a TMDnucleotide sequence selected from the group of: (a) a nucleotidesequence of a polynucleotide sequence selected from any of SEQ IDNOS:1–21 and 95–142, wherein said nucleotide sequence encodes a TMDpolypeptide; (b) a nucleotide sequence that encodes the TMD of apolypeptide selected from any of SEQ ID NOS:48–68, 174–221, 283–285, and290–293; and (c) a nucleotide sequence which, but for codon degeneracy,hybridizes under at least stringent or highly stringent conditions oversubstantially the entire length of a nucleotide sequence (a) or (b).

Some such isolated or recombinant polypeptides further comprise acytoplasmic domain (CD) amino acid sequence encoded by a CD nucleotidesequence selected from the group of: (a) a nucleotide sequence of apolynucleotide sequence selected from any of SEQ ID NOS:1–21 and 95–142,wherein said nucleotide sequence encodes a CD polypeptide; (b) anucleotide sequence that encodes the CD of a polypeptide selected fromany of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293; and (c) anucleotide sequence which, but for codon degeneracy, hybridizes under atleast stringent or highly stringent conditions over substantially theentire length of a nucleotide sequence (a) or (b).

For some of the CD28BP nucleic acids described above, the polypeptideencoded by the nucleic acid has one of more of the followingproperties: 1) a CD28/CTLA-4 binding affinity ratio equal to, aboutequal to, or greater than the CD28/CTLA-4 binding affinity ratio ofhuman B7-1; 2) either an equal or an enhanced binding affinity for CD28as compared to a binding affinity of a wild type co-stimulatory moleculefor CD28; 3) a decreased or a lowered binding affinity for CTLA-4 ascompared to a binding affinity of a wild type co-stimulatory moleculefor CTLA-4; induces T-cell proliferation or T-cell activation or both;or 4) modulates T-cell activation, but does not induce proliferation ofpurified T-cells activated by soluble anti-CD3 mAbs.

In another embodiment, the invention provides isolated or recombinantnucleic acids each comprising a nucleotide sequence selected from thegroup of: (a) a nucleotide sequence that encodes an extracellular domain(ECD), said nucleotide sequence comprising an ECD coding subsequence ofa polynucleotide sequence selected from the group of SEQ ID NOS:1–21 and95–142, or a complementary nucleotide sequence thereof; (b) a nucleotidesequence encoding an ECD, said ECD comprising an amino acid subsequenceof a polypeptide sequence selected from the group of SEQ ID NOS:48–68,174–221, 283–285, and 290–293, or a complementary nucleotide sequencethereof; and (c) a nucleotide sequence that, but for codon degeneracy,hybridizes under at least stringent conditions over substantially theentire length of polynucleotide sequence (a) or (b), wherein saidnucleotide sequence encodes a polypeptide that has a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than the CD28/CTLA-4 bindingaffinity ratio of human B7-1 or an ability to induce a T cellproliferation response about equal to or greater than that induced byhB7-1.

For some such isolated or recombinant nucleic acids, the nucleotidesequence of (c) hybridizes under at least stringent conditions oversubstantially the entire length of polynucleotide sequence (a) andencodes a polypeptide that has a CD28/CTLA-4 binding affinity ratiogreater than the CD28/CTLA-4 binding affinity ratio of human B7-1 orinduces a T cell proliferation response greater than that induced byhB7-1. Some such isolated or recombinant nucleic acids further compriseat least a second nucleotide sequence that encodes a signal peptide,wherein said second nucleotide sequence is selected from the group of:(a) a nucleotide sequence comprising a signal peptide coding subsequenceof a polynucleotide sequence selected from the group of SEQ ID NOS:1–21and 95–142, or a complementary nucleotide sequence thereof; (b) anucleotide sequence encoding a signal peptide, said signal peptidecomprising an amino acid subsequence of a polypeptide sequence selectedfrom the group of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, or acomplementary nucleotide sequence thereof; (c) a nucleotide sequencethat, but for codon degeneracy, hybridizes under at least stringentconditions over substantially the entire length of polynucleotidesequence (a) or (b), wherein said nucleotide sequence encodes apolypeptide that has a CD28/CTLA-4 binding affinity ratio about equal toor greater than that of hB7-1, or an ability to induce a T cellproliferation or activation response about equal to or greater than thatinduced by hB7-1; and (d) a nucleotide sequence encoding a signalpeptide of a B7-1 polypeptide.

For some such isolated or recombinant nucleic acids, the nucleotidesequence of (c) hybridizes, but for codon degeneracy, under at leaststringent conditions over substantially the entire length ofpolynucleotide sequence (a) and encodes a polypeptide that has aCD28/CTLA-4 binding affinity ratio greater than that of hB7-1 or anability to induce a T cell proliferation response about equal to orgreater than that induced by hB7-1. Some such nucleic acids furthercomprise at least a third nucleotide sequence encoding a transmembranedomain selected from the group of: (a) a nucleotide sequence comprisinga transmembrane domain coding subsequence of a polynucleotide sequenceselected from the group of SEQ ID NOS:1–21 and 95–142, or acomplementary nucleotide sequence thereof; (b) a nucleotide sequenceencoding a transmembrane domain, said transmembrane domain comprising anamino acid subsequence of a polypeptide sequence selected from the groupof SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, or a complementarynucleotide sequence thereof; (c) a nucleotide sequence that hybridizes,but for the degeneracy of the genetic code, under at least stringentconditions over substantially the entire length of polynucleotidesequence (a) or (b), wherein said nucleotide sequence encodes apolypeptide that has a CD28/CTLA-4 binding affinity ratio about equal toor greater than that of hB7-1; and (d) a nucleotide sequence thatencodes a transmembrane domain of a B7-1 polypeptide.

Further, some such nucleic acids further comprise at least a fourthnucleotide sequence encoding a cytoplasmic domain selected from thegroup of: (a) a nucleotide sequence comprising a cytoplasmic domaincoding subsequence of a polynucleotide sequence selected from the groupof SEQ ID NOS:1–21 and 95–142, or a complementary nucleotide sequencethereof; (b) a nucleotide sequence encoding a cytoplasmic domain, saidcytoplasmic domain comprising an amino acid subsequence of a polypeptidesequence selected from the group of SEQ ID NOS:48–68, 174–221, 283–285,and 290–293, or a complementary nucleotide sequence thereof; (c) anucleotide sequence that, but for codon degeneracy, hybridizes under atleast stringent conditions over substantially the entire length ofpolynucleotide sequence (a) or (b), wherein said nucleotide sequenceencodes a polypeptide that has a CD28/CTLA-4 binding affinity ratioabout equal to or greater than that ratio of human B7-1; and (d) anucleotide sequence that encodes a cytoplasmic domain of a B7-1polypeptide.

CTLA-4BP Polynucleotides

The invention includes isolated or recombinant nucleic acids that eachcomprise a polynucleotide sequence selected from: (a) a polynucleotidesequence selected from SEQ ID NOS:22–45, 143–173, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:69–92, 222–247, 286–289, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which, but for codon degeneracy, hybridizes under at leaststringent or highly stringent conditions over substantially the entirefull-length length of polynucleotide sequence (a) or (b); and (d) apolynucleotide sequence comprising all or a fragment of (a), (b), or(c); wherein (c) or (d) encodes a polypeptide having anaturally-occurring or non naturally-occurring sequence comprising atleast one of: Gly at position 2; Thr at position 4; Arg at position 5;Gly at position 8; Pro at position 12; Met at position 25; Cys atposition 27; Pro at position 29; Leu at position 31; Arg at position 40;Leu at position 52; His at position 65; Ser at position 78; Asp atposition 80; Tyr at position 87; Lys at position 120; Asp at position122; Lys at position 129; Met at position 135; Phe at position 150; Ileat position 160; Ala at position 164; His at position 172; Phe atposition 174; Leu at position 176; Asn at position 178; Asn at position186; Glu at position 194; Gly at position 196; Thr at position 199; Alaat position 210; His at position 212; Arg at position 219; Pro atposition 234; Asn at position 241; Leu at position 244; Thr at position250; Ala at position 254; Tyr at position 265; Arg at position 266; Gluat position 273; Lys at position 275; Ser at position 276; an amino aciddeletion at position 276; and Thr at position 279, wherein the positionnumber corresponds to that of the human B7-1 amino acid sequence (SEQ IDNO:278), and wherein said polypeptide has a CTLA-4/CD28 binding affinityratio about equal to, equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of human B7-1.

In another aspect, the invention provides isolated or recombinantnucleic acids that comprise a polynucleotide sequence selected from: (a)a polynucleotide sequence selected from SEQ ID NOS:253–262, or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence encoding a polypeptide selected from SEQ ID NOS:263–272, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which, but for codon degeneracy, hybridizes under highlystringent conditions over substantially the entire length ofpolynucleotide sequence (a) or (b) and encodes a polypeptide having anon naturally-occurring sequence; and (d) a polynucleotide sequencecomprising all or a fragment of (a), (b), or (c), wherein the fragmentencodes a polypeptide having (i) a naturally-occurring or nonnaturally-occurring sequence and (ii) a CTLA-4/CD28 binding affinityratio about equal to or greater than that of human B7-1.

In another aspect, the invention provides isolated or recombinantnucleic acids comprising a polynucleotide sequence encoding apolypeptide, the encoded polypeptide comprising an amino acid sequencewhich is substantially identical over at least about 125, 150, 175, 200,225, 250, or more contiguous amino acid residues of any one of SEQ IDNOS:69–92, 222–247, 263–272, and 286–289.

The invention also provides isolated or recombinant nucleic acids thateach comprise a nucleotide sequence coding for a polypeptide comprisingthe amino acid sequence set forth in any of SEQ ID NOS:69–92, 222–247,263–272, and 286–289, or a subsequence thereof, wherein the subsequencecomprises at least one of: the signal sequence, extracellular domain,transmembrane domain, and cytoplasmic domain of said polypeptide, andwherein the amino acid sequence or subsequence is a nonnaturally-occurring sequence.

For some such CTLA-4BP nucleic acids described above, a polypeptideencoded therefrom has a CTLA-4/CD28 binding affinity ratio about equalto, equal to or greater than the CTLA-4/CD28 binding affinity ratio ofhuman B7-1. Furthermore, the polypeptide encoded by some such CTLA-4BPnucleic acids has either a same binding affinity or an enhanced bindingaffinity for CD28 as compared to a binding affinity of a wild typeco-stimulatory molecule for CD28. Some such encoded polypeptides have adecreased or a lowered binding affinity for CTLA-4 as compared to abinding affinity of a wild type co-stimulatory molecule for CTLA-4(e.g., a mammalian B7-1, such as hB7-1). Some such encoded polypeptidesinhibit either or both T-cell proliferation or T-cell activation. Somesuch encoded polypeptides modulate T-cell activation, but do not induceproliferation of purified T-cells activated by soluble anti-CD3 mAbs.

In addition, the invention provides novel isolated or recombinantnucleic acids corresponding to baboon and orangutan B7-1. Such sequencescomprise a polynucleotide sequence selected from: (a) a polynucleotidesequence selected from SEQ ID NO:46, SEQ ID NO:47, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NO:93, SEQ ID NO:94, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence encoding a subsequence of a polypeptide selected from SEQ IDNO:93, SEQ ID NO:94, or a complementary polynucleotide sequence thereof,wherein the subsequence comprises at least one of: the signal sequence,extracellular domain, transmembrane domain, and the cytoplasmic domainof said polypeptide. Included are polypeptide fragments (e.g., 100, 150,200, or 250 amino acids) and nucleotides encoding such fragments, havingCD28 and/or CTLA-4 binding properties similar or about equal to those ofhB7-1 and/or an ability to induce T cell proliferation or activationresponse similar or about equal to that of hB7-1.

Additional Aspects

The invention also includes RNA sequences that correspond to each of theNCSM DNA sequences of the invention. For example, included is an RNAsequence comprising the NCSM DNA sequence of any of SEQ ID NOS:1–47,95–173, and 253–262, wherein a uracil residue is substituted for eachthymidine residue in said DNA sequence, and a complementary sequence ofeach such RNA sequence. Also included is a RNA sequence comprising anucleotide sequence comprising at least one of an ECD coding sequence,TMD coding sequence, CD coding nucleotide sequence, and/or signalpeptide coding nucleotide sequence of DNA sequence of any of SEQ IDNOS:1–47, 95–173, and 253–262, wherein a uracil residue is substitutedfor each thymidine residue in said DNA sequence, and complementarysequences of such RNA sequence. The DNA subsequence of SEQ ID NOS:1–47,95–173, and 253–262, that encodes each of the ECD, TMD, CD, and signalpeptide are readily determined by alignment with the DNA sequence ofhB7-1 (see, e.g., FIGS. 2 and 3). The invention further provides a viruscomprising a nucleic acid or polynucleotide (RNA or DNA) of theinvention.

Any of the CD28BP and CTLA-4BP nucleic acids described above may acidencode a fusion protein comprising at least one additional amino acidsequence. The at least one additional amino acid sequence comprises anIg polypeptide. The polypeptide may comprise a human IgG polypeptide orFc domain of an IgG polypeptide, and may comprise an Fc hinge, a CH2domain, and a CH3 domain. Exemplary IgG1 polypeptides and theirsequences are shown in the Examples below.

A polypeptide encoded by any of the CD28BP and CTLA-4BP nucleic acidsdescribed above may comprise at least one of a signal sequence, aprecursor peptide, and an epitope tag sequence or Histidine tag.

In another aspect, the invention provides cells comprising one or moreof the CD28BP or CTLA-4BP nucleic acids described above. Such cells mayexpress one or more polypeptides encoded by the nucleic acids of theinvention.

The invention also provides vectors comprising any of the CTLA-4BP orCD28BP nucleic acids described above. Such vectors may comprise aplasmid, a cosmid, a phage, a virus, or a fragment of a virus. Suchvectors may comprise an expression vector, and, if desired, the CD28BPor CTLA-4BP nucleic acid is operably linked to a promoter, includingthose discussed herein and below.

Such a vector may be a bicistronic vector, comprising in addition to anucleotide sequence encoding a CD28BP or CTLA-4BP, a nucleotide sequenceencoding a transgene, such as an antigen, marker, or otherco-stimulatory molecule, or cytokine (e.g., GM-CSF, IL-12, or IL-2).Such vector may also be tricistronic or of higher order, comprising afurther (third) nucleotide acid sequence. For example, the vector maycomprise nucleotide sequences encoding an NCSM polypeptide (e.g., CD28BPor CTLA-4BP), antigen, and cytokine, such as IL-12. In one embodiment,the antigen is a cancer antigen, such as EpCam (or mutant or variantpolypeptide thereof) or another cancer antigen described below, or viralantigen. In such expression vector, the nucleic acid may be operablylinked to first promoter and the polynucleotide sequence encoding theantigen may operably linked to a second promoter. Each promoter cancomprise any promoter described below. In one aspect, one or bothpromoters in the expression vector that includes a CD28BP or CTLA-4BPpolypeptide-encoding nucleotide sequence is a CMV promoter or variantthereof. The vector may further comprise a bovine growth hormone (BGH)poly adenylation sequence or SV40 polyA sequence.

A preferred “backbone” expression vector is that shown in FIG. 21; theexpression vector components shown in this backbone vector may be usedwith any NCSM nucleic acid sequence. Other expression vector elementsthat can be employed and other vector types and formats are described indetail below. A preferred expression vector that includes a CD28BP orCTLA-4BP polypeptide-encoding nucleotide sequence is shown in FIG. 22A.The components of a preferred bicistronic expression vector thatincludes a CD28BP polypeptide-encoding nucleotide sequence, such as thatencoding clone CD28BP-15, and a nucleic acid sequence encoding EpCam areshown in FIG. 23A.

The invention also provides host cells comprising any of the vectorsthat comprise nucleotide sequences encoding any CD28BP or CTLA-4BPdescribed herein.

Furthermore, in another aspect, the invention provides compositionscomprising an excipient or carrier and at least one of any of the CD28BPor CTLA-4BP nucleic acids, or vectors, cells, or host comprising suchnucleic acids. Such composition may be pharmaceutical compositions, andthe excipient or carrier may be a pharmaceutically acceptable excipientor carrier.

The invention also includes compositions comprising two or more NCSMpolynucleotides of the invention or fragments thereof (e.g., assubstrates for recombination). The composition can comprise a library ofrecombinant nucleic acids, where the library contains at least 2, atleast 3, at least 5, at least 10, at least 20, at least 50, or at least100 or more nucleic acids described above. The nucleic acids areoptionally cloned into expression vectors, providing expressionlibraries.

The NCSM polynucleotides of the invention and fragments thereof, as wellas vectors comprising such polynucleotides, may be employed fortherapeutic or prophylactic uses in combination with a suitable carrier,such as a pharmaceutical carrier. Such compositions comprise atherapeutically and/or prophylactically effective amount of thecompound, and a pharmaceutically acceptable carrier or excipient. Such acarrier or excipient includes, but is not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The formulation should suit the mode of administration. Methods ofadministering nucleic acids, polypeptides, and proteins are well knownin the art, and are further discussed below.

The invention also includes compositions produced by digesting one ormore of any of the NCSM nucleic acids described above with a restrictionendonuclease, an RNAse, or a DNAse (e.g., as is performed in certain ofthe recombination formats noted above); and compositions produced byfragmenting or shearing one or more NCSM polynucleotides of theinvention by mechanical means (e.g., sonication, vortexing, and thelike), which can also be used to provide substrates for recombination inthe methods described herein. The invention also provides compositionsproduced by cleaving at least one of any of the CD28BP or CTLA-4BPnucleic acids described above. The cleaving may comprise mechanical,chemical, or enzymatic cleavage, and the enzymatic cleavage may comprisecleavage with a restriction endonuclease, an RNAse, or a DNAse.

Also included in the invention are compositions produced by a processcomprising incubating one or more of the fragmented nucleic acid sets inthe presence of ribonucleotide or deoxyribonucleotide triphosphates anda nucleic acid polymerase. This resulting composition forms arecombination mixture for many of the recombination formats noted above.The nucleic acid polymerase may be an RNA polymerase, a DNA polymerase,or an RNA-directed DNA polymerase (e.g., a “reverse transcriptase”); thepolymerase can be, e.g., a thermostable DNA polymerase (e.g., VENT, TAQ,or the like).

Similarly, compositions comprising sets of oligonucleotidescorresponding to more than one NCSM nucleic acids of the invention areuseful as recombination substrates and are a feature of the invention.For convenience, these fragmented, sheared, or oligonucleotidesynthesized mixtures are referred to as fragmented nucleic acid sets.

In one aspect, the invention provides an isolated or recombinant nucleicacid encoding a polypeptide that has a CTLA-4/CD28 binding affinityratio about equal to or greater than the CTLA-4/CD28 binding affinityratio of hB7-1, produced by mutating or recombining at least oneCTLA-4BP nucleic acid described above. In another aspect, the inventionprovides an isolated or recombinant nucleic acid encoding a polypeptidethat has a CD28/CTLA-4 binding affinity ratio about equal to or greaterthan the CD28/CTLA-4 binding affinity ratio of hB7-1, produced bymutating or recombining at least one CD28BP nucleic acid describedabove.

The invention also provides a chimeric or recombinant polynucleotidethat encodes a polypeptide having a CD28/CTLA-4 binding affinity ratioabout equal to or greater than the CD28/CTLA-4 binding affinity ratio ofhB7-1. In some aspects, such encoded polypeptide is a mammalian B7-1variant. In some aspects, such polypeptide comprises an amino acidsequence comprising one or more amino acid subsequences corresponding toamino acid subsequences of wild-type cow B7-1, baboon B7-1, rabbit B7-1,and human B7-1 polypeptides. In some aspects, such polypeptide exhibitsan ability to induce a T cell proliferation or activation response of Tcells (e.g., stimulated by anti-CD3 Abs or antigen) greater than that ofcow B7-1, rabbit B7-1 or human B7-1. Chimeric or recombinantpolypeptides encoded therefrom are also an aspect of the invention (see,e.g., FIG. 8B).

In addition, the invention includes a chimeric or recombinantpolynucleotide that encodes a polypeptide having a CTLA-4/CD28 bindingaffinity ratio about equal to or greater than that of hB7-1. In someaspects, such encoded polypeptide is a mammalian B7-1 variant. In someaspects, such polypeptide comprises an amino acid sequence comprisingone or more amino acid subsequences corresponding to amino acidsubsequences of wild-type rhesus B7-1, baboon B7-1, human B7-1,orangutan B7-1, and cow B7-1 polypeptides. In some aspects, suchpolypeptide exhibits an ability to suppress or inhibit a T cellproliferation or activation response (e.g., of T cells stimulated byanti-CD3 Abs or antigen) relative to that induced human B7-1. Chimericor recombinant polypeptides encoded therefrom are also an aspect of theinvention (see, e.g., FIG. 8A).

Making Polynucleotides

NCSM polynucleotides, oligonucleotides, and nucleic acid fragments ofthe invention can be prepared by standard solid-phase methods, accordingto known synthetic methods. Typically, fragments of up to about 100bases are individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous sequence. For example, theNCSM polynucleotides and oligonucleotides of the invention can beprepared by chemical synthesis using, e.g., classical phosphoramiditemethod described by, e.g., Beaucage et al. (1981) Tetrahedron Letters22:1859–69, or the method described by Matthes et al. (1984) EMBO J3:801–05, e.g., as is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned into appropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company (mcrc@oligos.com), The Great American Gene Company(http://www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.) and many others. Similarly, peptidesand antibodies can be custom ordered from any of a variety of sources,e.g., PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.(http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis,Inc., and many others.

Certain NCSM polynucleotides of the invention may also be obtained byscreening cDNA libraries (e.g., libraries generated by recombininghomologous nucleic acids as in typical recursive sequence recombinationmethods) using oligonucleotide probes that can hybridize to orPCR-amplify polynucleotides which encode the NCSM polypeptides andfragments of those polypeptides. Procedures for screening and isolatingcDNA clones are well-known to those of skill in the art. Such techniquesare described in, e.g., Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymol. Vol. 152, Acad. Press, Inc., San Diego,Calif. (“Berger”); Sambrook, supra, and Current Protocols in MolecularBiology, Ausubel, supra. Some NCSM polynucleotides of the invention canbe obtained by altering a naturally occurring backbone, e.g., bymutagenesis, recursive sequence recombination (e.g., shuffling), oroligonucleotide recombination. In other cases, such polynucleotides canbe made in silico or through oligonucleotide recombination methods asdescribed in the references cited herein.

As described in more detail herein, the NCSM polynucleotides of theinvention include polynucleotide sequences that encode NCSM polypeptidesequences and fragments thereof (including all forms of soluble NCSMpolypeptides and fusion proteins), polynucleotide sequencescomplementary to these polynucleotide sequences and fragments thereof,polynucleotides that hybridize under at least stringent conditions toNCSM sequences defined herein, novel fragments of coding sequences andcomplementary sequences thereof, and variants, analogs, and homologuederivatives of all of the above. A coding sequence refers to anucleotide sequence encodes a particular polypeptide or domain, region,or fragment of said polypeptide. A coding sequence may code for a NCSMpolypeptide or fragment thereof having a functional property, such as aan ability to bind a receptor, induce or suppress T cell proliferationin conjunction with stimulation of T cell receptor (by, e.g., an antigenor anti-CD3 Ab), or induce or stimulate a cytokine response as describedherein. The polynucleotides of the invention can be in the form of RNAor in the form of DNA, and include mRNA, cRNA, synthetic RNA and DNA,and cDNA. The polynucleotides can be double-stranded or single-stranded,and if single-stranded, can be the coding strand or the non-coding(anti-sense, complementary) strand. The NCSM polynucleotides optionallyinclude the coding sequence of a NCSM polypeptide (i) in isolation, (ii)in combination with one or more additional coding sequences, so as toencode, e.g., a fusion protein, a pre-protein, a prepro-protein, or thelike, (iii) in combination with non-coding sequences, such as introns,control elements, such as a promoter (e.g., naturally occurring orrecombinant or shuffled promoter), a terminator element, or 5′ and/or 3′untranslated regions effective for expression of the coding sequence ina suitable host, and/or (iv) in a vector, cell, or host environment inwhich NCSM coding sequence is a heterologous gene. The NCSMpolynucleotides include the respective coding sequences of components ofa NCSM polypeptide, including, e.g., the coding sequence for each of thesignal peptide, ECD, transmembrane domain, cytoplasmic domain, matureregion, and fragments thereof, and variants, analogs, and homologuederivatives thereof. Polynucleotide sequences can also be found incombination with typical compositional formulations of nucleic acids,including in the presence of carriers, buffers, adjuvants, excipients,and the like, as are known to those of ordinary skill in the art. NCSMnucleotide fragments typically comprise at least about 500 nucleotidebases, usually at least about 600, 650, or 700 bases, and often 750 ormore bases. The nucleotide fragments, variants, analogs, and homologuederivatives of NCSM polynucleotides may have hybridize under highlystringent conditions to a NCSM polynucleotide or homologue sequencedescribed herein and/or encode amino acid sequences having at least oneof the properties of receptor binding, ability to alter an immuneresponse via, e.g., T cell activation /proliferation, and cytokineproduction of NCSM polypeptides described herein.

Using Polynucleotides

The NCSM polynucleotides and fragments, variants, and homologues thereofof the invention have a variety of uses in, for example, recombinantproduction (i.e., expression) of the NCSM polypeptides of the inventiontypically through expression of a plasmid expression vector comprising asequence encoding a NCSM polypeptide or fragment thereof (e.g., ECDdomain); as therapeutics; as prophylactics; as diagnostic tools; asimmunogens; as adjuvants; as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of natural B7-1 or related co-stimulatory molecule codingnucleic acids) as substrates for further reactions, e.g., recursivesequence recombination reactions or mutation reactions to produce newand/or improved homologues, and the like. Such NCSM polynucleotides andfragments, variants, and homologues thereof of the invention can beadministered to a subject by any one of the delivery routes describedbelow (including, but not limited to, e.g., intramuscularly,intradermally, subdermally, subcutaneously, orally, intraperitoneally,intrathecally, intravenously, mucosally, systemically, parenterally, viainhalation, or placed within a cavity of the body (including, e.g.,during surgery)).

Expression of Polypeptides from Polynucleotides

In accordance with the present invention, NCSM polynucleotide sequenceswhich encode novel full-length or mature NCSM polypeptides or proteins,fragments, variants or homologues thereof, related fusion polypeptidesor proteins, or functional equivalents thereof, collectively referred toherein, e.g., as “NCSM” molecules, are used in recombinant DNA moleculesthat direct the expression of the NCSM polypeptides in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other nucleicacid sequences that encode substantially the same or a functionallyequivalent amino acid sequence are also used to synthesize, clone andexpress the NCSM polypeptides.

Modified Coding Sequences

As will be understood by those of ordinary skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms preferentially use a subset of these codons. Thecodons that are utilized most often in a species are called optimalcodons, and those not utilized very often are classified as rare orlow-usage codons (see, e.g., Zhang, S. P. et al. (1991) Gene 105:61–72).Codons can be substituted to reflect the preferred codon usage of thehost, a process called “codon optimization” or “controlling for speciescodon bias.”

Optimized coding sequence containing codons preferred by a particularprokaryotic or eukaryotic host (see, e.g., Murray, E. et al. (1989) NucAcids Res 17:477–508) can be prepared, for example, to increase the rateof translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,preferred stop codons for S. cerevisiae and mammals are UAA and UGArespectively. The preferred stop codon for monocotyledonous plants isUGA, whereas insects and E. coli prefer to use UAA as the stop codon(Dalphin, M. E. et al. (1996) Nuc Acids Res 24:216–218).

The polynucleotide sequences of the present invention can be engineeredin order to alter an NCSM coding sequence of the invention for a varietyof reasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the gene product. For example,alterations may be introduced using techniques which are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, to alter glycosylation and/or pegylation patterns, to changecodon preference, to introduce splice sites, etc. Further detailsregarding silent and conservative substitutions are provided below.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the nucleic acid sequences as broadly described above.The constructs comprise a vector, such as, a plasmid, a cosmid, a phage,a virus, a virus-like particle, a bacterial artificial chromosome (BAC),a yeast artificial chromosome (YAC), and the like, into which a nucleicacid sequence of the invention (e.g., one which encodes a NCSMpolypeptide or fragment thereof) has been inserted, in a forward orreverse orientation. In a preferred aspect of this embodiment, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the nucleic acid sequence. Largenumbers of suitable vectors and promoters are known to those of skill inthe art, and are commercially available.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger, supra; Sambrook (1989), supra, and Ausubel,supra. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, all supra, as wellas Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guideto Methods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36–47; The Journal Of NIH Research (1991) 3:81–94; (Kwoh et al. (1989)Proc Natl Acad Sci USA 86:1173–1177; Guatelli et al. (1990) Proc NatlAcad Sci USA 87:1874–1878; Lomeli et al. (1989) J Clin Chem35:1826–1831; Landegren et al. (1988) Science 241:1077–1080; Van Brunt(1990) Biotechnology 8:291–294; Wu and Wallace (1989) Gene 4:560–569;Barringer et al. (1990) Gene 89:117–122, and Sooknanan and Malek (1995)Biotechnology 13:563–564. Improved methods of cloning in vitro amplifiednucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.Improved methods of amplifying large nucleic acids by PCR are summarizedin Cheng et al. (1994) Nature 369:684–685 and the references therein, inwhich PCR amplicons of up to 40 kilobases (kb) are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. SeeAusubel, Sambrook and Berger, all supra.

The present invention also provides host cells that are transduced withvectors of the invention, and the production of polypeptides of theinvention by recombinant techniques. Host cells are geneticallyengineered (e.g., transduced, transformed or transfected) with thevectors of this invention, which may be, for example, a cloning vectoror an expression vector. The vector may be, for example, in the form ofa plasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants, or amplifying the NCSMgene. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to those skilled in the art and in the references citedherein, including, e.g., Freshney (1994) Culture of Animal Cells, aManual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein.

The NCSM polypeptides of the invention can also be produced innon-animal cells such as plants, yeast, fungi, bacteria and the like. Inaddition to Sambrook, Berger and Ausubel, details regarding cell cultureare found in, e.g., Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.);Atlas & Parks (eds.) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fla.

The polynucleotides of the present invention and fragments and variantsthereof, which encode the NCSM polypeptide molecules, may be included inany one of a variety of expression vectors for expressing a polypeptide.Such vectors include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40, bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adeno-associated virus, retroviruses and manyothers. Any vector that transduces genetic material into a cell, and, ifreplication is desired, which is replicable and viable in the relevanthost can be used.

The nucleic acid sequence in the expression vector is operatively linkedto an appropriate transcription control sequence (promoter) to directmRNA synthesis. Examples of such promoters include: LTR or SV40promoter, E. coli lac or trp promoter, phage lambda P_(L) promoter, CMVpromoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. The expression vectoralso contains a ribosome binding site for translation initiation, and atranscription terminator. The vector optionally includes appropriatesequences for amplifying expression, e.g., an enhancer. In addition, theexpression vectors optionally comprise one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or such as tetracycline or ampicillinresistance in E. coli.

The vector containing the appropriate DNA sequence encoding a NCSMpolypeptide, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. Examples of appropriate expression hosts include:bacterial cells, such as E. coli, Streptomyces, and Salmonellatyphimurium; fungal cells, such as Saccharomyces cerevisiae, Pichiapastoris, and Neurospora crassa; insect cells such as Drosophila andSpodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 orBowes melanoma; plant cells, etc. It is understood that not all cells orcell lines need to be capable of producing fully functional NCSMpolypeptides or fragments thereof; for example, antigenic fragments ofNCSM polypeptide may be produced in a bacterial or other expressionsystem. The invention is not limited by the host cells employed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the NCSM polypeptide or fragmentthereof. For example, when large quantities of a NCSM polypeptide orfragments thereof are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which NCSM nucleotide coding sequence may beligated into the vector in-frame with sequences for the amino-terminalMet and the subsequent 7 residues of beta-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503–5509); pET vectors (Novagen, Madison Wis.); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of the NCSMpolypeptides of the invention. For reviews, see Ausubel, supra, Berger,supra, and Grant et al. (1987) Methods in Enzymology 153:516–544.

In mammalian host cells, a number of expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome results in a viablevirus capable of expressing NCSM molecule in infected host cells (Loganand Shenk (1984) Proc Natl Acad Sci USA 81:3655–3659). In addition,transcription enhancers, such as the rous sarcoma virus (RSV) enhancer,are used to increase expression in mammalian host cells. Host cells,media, expression systems, and methods of production include those knownfor cloning and expression of various mammalian B7-1s (e.g., hB7-1 andmouse B7-1).

Promoters for use with NCSM polynucleotide sequences of the presentinvention include recombinant, mutated, or recursively recombined (e.g.,shuffled) promoters, including optimized recombinant CMV promoters, asdescribed in copending, commonly assigned PCT Application Serial No. US01/20123, entitled “Novel Chimeric Promoters,” filed Jun. 21, 2001,incorporated herein by reference in its entirety for all purposes. Suchpromoters can be employed in expression vectors comprising nucleotidesequences encoding, e.g., NCSM polypeptides, soluble NSCM-ECDpolypeptides, or NCSM-ECD-Ig fusion proteins, or WT hB7-1, or fragmentsof any of these.

In some embodiments, a recombinant or shuffled promoter having anoptimized expression for a particular use with NCSM molecules isutilized. For example, in some therapeutic and/or prophylactic methodsor applications, where a lower level expression of a CD28BP or CTLA-4BPis desired (than is typically obtained with a CMV promoter, such as a WThuman CMV promoter), at least one recombinant or chimeric CMV promoternucleotide sequence that is optimized to provide for reduced orsuppressed expression levels of the NCSM and/or one or more associatedantigens is used. Such promoter(s) is operably linked in an expressionvector to either or both the NCSM polynucleotide and/or one or moreassociated antigens (e.g., cancer antigen, such as EpCam/KSA or mutant,variant or derivative of EpCam/KSA). In other embodiments, one or morerecombinant, mutant, or chimeric CMV promoters optimized for theparticular application can be used, where differential expressionbetween a NCSM polypeptide and at least one associated antigen in one ormore vectors is desired (e.g., where it is desirable to express varyingamounts of various NCSM polypeptide molecules or co-stimulatorymolecules, since their respective concentrations influence or affect oneanother, and/or where it is desirable to express a comparably higherlevel of at least one antigen for effective treatment). For example, insome applications, a low expression level of a NCSM polypeptide and arelatively higher expression level of antigen is desired, since it maybe particularly useful for successful therapeutic or prophylactictreatment of a particular condition or disease.

Additional Expression Elements

Specific initiation signals can aid in efficient translation of a NCSMpolynucleotide coding sequence and/or fragments thereof. These signalscan include, e.g., the ATG initiation codon and adjacent sequences. Incases where a NCSM coding sequence, its initiation codon and upstreamsequences are inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only coding sequence (e.g., a mature protein codingsequence), or a portion thereof, is inserted, exogenous nucleic acidtranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression canenhanced by the inclusion of enhancers appropriate to the cell system inuse (see, e.g., Scharf D. et al. (1994) Results Probl Cell Differ20:125–62; and Bittner et al. (1987) Methods in Enzymol 153:516–544).

Secretion/Localization Sequences

Polynucleotides of the invention encoding NCSM polypeptides andfragments thereof can also be fused, for example, in-frame to nucleicacid encoding a secretion/localization sequence, to target polypeptideexpression to a desired cellular compartment, membrane, or organelle, orto direct polypeptide secretion to the periplasmic space or into thecell culture media. Such sequences are known to those of skill, andinclude secretion leader or signal peptides, organelle targetingsequences (e.g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e.g., stop transfer sequences,GPI anchor sequences), and the like.

Expression Hosts

In a further embodiment, the present invention relates to host cellscontaining any of the above-described nucleic acids, vectors, or otherconstructs of the invention. The host cell can be a eukaryotic cell,such as a mammalian cell, a yeast cell, or a plant cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the construct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, geneor vaccine gun, injection, or other common techniques (see, e.g., Davis,L., Dibner, M., and Battey, I. (1986) Basic Methods in MolecularBiology) for in vivo, ex vivo or in vitro methods.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, pegylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “pre” or a “prepro” formof the protein may also be important for correct insertion, foldingand/or function. Different host cells such as E. coli, Bacillus sp.,yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38,etc. have specific cellular machinery and characteristic mechanisms forsuch post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression can be used. For example, cell lines which stably express apolypeptide of the invention are transduced using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1–2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced sequences. For example, resistant clumps of stablytransformed cells can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a NCSMpolypeptide or fragments thereof of the invention are optionallycultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein or fragment thereofproduced by a recombinant cell may be secreted, membrane-bound, orcontained intracellularly, depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing polynucleotides encoding mature NCSM polypeptides ofthe invention can be designed with signal sequences which directsecretion of the mature polypeptides through a prokaryotic or eukaryoticcell membrane.

The present invention also includes at least one NCSM polynucleotideconsensus sequence derived from a comparison of two or more NCSMpolynucleotide sequences described herein (including, e.g., apolynucleotide encoding a CD28BP or CTLA-4BP of the invention orfragment (e.g., ECD or trunECD) thereof). The present invention alsoincludes at least one NCSM polynucleotide consensus sequence derivedfrom a comparison of two or more NCSM polynucleotide sequences describedherein. A NCSM polynucleotide consensus sequence as used herein means anonnaturally-occurring or recombinant NCSM polynucleotide sequence thatpredominantly includes those nucleic acid residues that are common toall NCSM polynucleotides of the present invention described herein andthat includes, at one or more of those positions wherein there is nonucleic acid residue common to all subtypes, a nucleic acid residue thatpredominantly occurs at that position and in no event includes anynucleic acid residue which is not extant in that position in at leastone NCSM polynucleotide of the invention.

Additional Sequences

The NCSM polypeptide-encoding polynucleotides of the present inventionoptionally comprise a coding sequence or fragment thereof fused in-frameto a marker sequence which, e.g., facilitates purification of theencoded polypeptide. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, a sequence which binds glutathione (e.g., GST), a hemagglutinin(HA) tag (corresponding to an epitope derived from the influenzahemagglutinin protein; Wilson, I. et al. (1984) Cell 37:767), maltosebinding protein sequences, the FLAG epitope utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle, Wash.),and the like. The inclusion of a protease-cleavable polypeptide linkersequence between the purification domain and the NCSM sequence is usefulto facilitate purification.

For example, one expression vector possible to use in the compositionsand methods described herein provides for expression of a fusion proteincomprising a polypeptide of the invention fused to a polyhistidineregion separated by an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography, as described in Porath et al. (1992) ProteinExpression and Purification 3:263–281) while the enterokinase cleavagesite provides a method for separating the NCSM polypeptide from thefusion protein. pGEX vectors (Promega; Madison, Wis.) are optionallyused to express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption to ligand-agarosebeads (e.g., glutathione-agarose in the case of GST-fusions) followed byelution in the presence of free ligand.

An additional construction in the compositions and methods describedherein provides for soluble proteins, and their encoding nucleic acids,comprising NCSM polypeptides (or one or more fragments thereof), e.g.,as described herein fused to an Ig molecule, e.g., human IgG Fc(“fragment crystallizable,” or fragment complement binding) hinge, CH2domain and CH3 domain (and nucleotide sequences encoding them). Fc isthe portion of the antibody responsible for binding to antibodyreceptors on cells and the C1q component of complement. Also includedare soluble forms of the NCSM polypeptides that comprise secreted formsof the NSCM polypeptides, as produced by chemical synthesis or, e.g., byintroducing a plasmid encoding a secreted form of the NCSM polypeptideinto a eukaryotic cell. These expressed or secreted soluble NCSMpolypeptides or fragments thereof, as well as the soluble NCSM fusionproteins (e.g., NCSM-ECD-Ig fusion proteins or NCSM-truncated-ECD-Igfusion proteins) or fragments thereof and their encoding nucleic acidsare optionally useful as prophylactic and/or therapeutic drugs or asdiagnostic tools (see also, e.g., Challita-Eid, P. et al. (1998) JImmunol 160:3419–3426; Sturmhoefel, K. et al. (1999) Cancer Res59:4964–4972).

Polypeptide Production and Recovery

Following transduction of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Eukaryotic or microbial cells employed in expression of the NCSMproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents, or other methods, which are well know to those skilled inthe art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See, e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W. H. Freeman and Company; andRicciardelli et al. (1989) In vitro Cell Dev Biol 25:1016–1024. Forplant cell culture and regeneration see, e.g., Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D.Croy (ed.) Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6.Cell culture media in general are set forth in Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue(1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and,e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature NCSM protein or fragments thereof. Finally, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted, supra, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ;Harris and Angal (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ.

In Vitro Expression Systems

Cell-free transcription/translation systems can also be employed toproduce NCSM polypeptides or fragments thereof using DNAs or RNAs of thepresent invention or fragments thereof. Several such systems arecommercially available. A general guide to in vitro transcription andtranslation protocols is found in Tymms (1995) In vitro Transcriptionand Translation Protocols: Methods in Molecular Biology Volume 37,Garland Publishing, NY.

Modified Amino Acids

Polypeptides of the invention may contain one or more modified aminoacids. The presence of modified amino acids may be advantageous in, forexample, (a) increasing polypeptide serum half-life, (b) reducingpolypeptide antigenicity, or (c) increasing polypeptide storagestability. Amino acid(s) are modified, for example, co-translationallyor post-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means.

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEG-ylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols on CD-ROM Humana Press, Towata, N.J.

In Vivo Uses and Applications

Polynucleotides or fragments thereof that encode a NCSM polypeptide ofthe invention, or complements of the polynucleotides (e.g., antisense orribozyme molecules), are optionally administered to a cell to accomplisha therapeutically useful process or to express a therapeutically usefulproduct. These in vivo applications, including gene therapy, include amultitude of techniques by which gene expression may be altered incells. Such methods include, for instance, the introduction of genes forexpression of, e.g., therapeutically and/or prophylactically usefulpolypeptides, such as the NCSM polypeptides of the present invention orfragments thereof.

In Vivo Polypeptide Expression

Polynucleotides encoding NCSM polypeptides of the invention andfragments thereof are particularly useful for in vivo therapeuticapplications, using techniques well known to those skilled in the art.For example, cultured cells are engineered ex vivo with at least oneNCSM polynucleotide (DNA or RNA) and/or other polynucleotide sequencesencoding, e.g., at least one of an antigen, cytokine, otherco-stimulatory molecule, adjuvant, etc., and the like, with theengineered cells then being returned to the patient. Cells may also beengineered in vivo for expression of one or more polypeptides in vivo.including NCSM polypeptides and/or antigenic peptides.

A number of viral vectors suitable for organismal in vivo transductionand expression are known. Such vectors include retroviral vectors (see,e.g., Miller, Curr Top Microbiol Immunol (1992) 158:1–24; Salmons andGunzburg (1993) Human Gene Therapy 4:129–141; Miller et al. (1994)Methods in Enzymology 217:581–599) and adeno-associated vectors(reviewed in Carter (1992) Curr Opinion Biotech 3:533–539; Muzcyzka(1992) Curr Top Microbiol Immunol. 158:97–129). Other viral vectors thatare used include adenoviral vectors, herpes viral vectors and Sindbisviral vectors, as generally described in, e.g., Jolly (1994) Cancer GeneTherapy 1:51–64; Latchman (1994) Molec Biotechnol 2:179–195; andJohanning et al. (1995) Nucl Acids Res 23:1495–1501.

In one aspect, a pox virus vector can be used. The pox viral vector istransfected with a polynucleotide sequence encoding of the NCSMpolypeptides (or fragments thereof) of the invention, such as a CD28BPpolypeptide, and is useful in prophylactic, therapeutic and diagnosticapplications where enhancement of an immune response, such as increasedor improved T cell proliferation or activation (or inhibition of animmune response, such as inhibition of T cell proliferation, if, e.g., apolynucleotide encoding a CTAL4-BP polypeptide is used) is desired. Seeviral vectors discussed in, e.g., Berencsi et al., J Infect Dis(2001)183(8):1171–9; Rosenwirth et al., Vaccine 2001 February8;19(13–14):1661–70; Kittlesen et al., J Immunol (2000) 164(8):4204–11;Brown et al. Gene Ther 2000 7(19):1680–9; Kanesa-thasan et al., Vaccine(2000) 19(4–5):483–91; Sten (2000) Drug 60(2):249–71. Compositionscomprising such vectors and an acceptable excipient are also a featureof the invention.

Gene therapy and genetic vaccines provide methods for combating chronicinfectious diseases (e.g., HIV infection, viral hepatitis), as well asnon-infectious diseases including cancer and some forms of congenitaldefects such as enzyme deficiencies, and such methods can be employedwith NCSM polynucleotides of the invention, including, e.g., vectors andcells comprising such polynucleotides. Several approaches forintroducing nucleic acids and vectors into cells in vivo, ex vivo and invitro have been used and can be employed with NCSM polynucleotidesencoding NCSM polypeptides and fragments thereof (including, e.g., ECDdomains and fusion proteins), and vectors comprising NCSM sequences.These approaches include liposome based gene delivery (Debs and Zhu(1993) WO 93/24640 and U.S. Pat. No. 5,641,662; Mannino andGould-Fogerite (1988) BioTechniques 6(7):682–691; Rose, U.S. Pat. No.5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) ProcNatl Acad Sci USA 84:7413–7414; Brigham et al. (1989) Am J Med Sci298:278–281; Nabel et al. (1990) Science 249:1285–1288; Hazinski et al.(1991) Am J Resp Cell Molec Biol 4:206–209; and Wang and Huang (1987)Proc Natl Acad Sci USA 84:7851–7855); adenoviral vector mediated genedelivery, e.g., to treat cancer (see, e.g., Chen et al. (1994) Proc NatlAcad Sci USA 91:3054–3057; Tong et al. (1996) Gynecol Oncol 61:175–179;Clayman et al. (1995) Cancer Res. 5:1–6; O'Malley et al. (1995) CancerRes 55:1080–1085; Hwang et al. (1995) Am J Respir Cell Mol Biol 13:7–16;Haddada et al. (1995) Curr Top Microbiol Immunol. 1995 (Pt. 3):297–306;Addison et al. (1995) Proc Natl Acad Sci USA 92:8522–8526; Colak et al.(1995) Brain Res 691:76–82; Crystal (1995) Science 270:404–410; Elshamiet al. (1996) Human Gene Ther 7:141–148; Vincent et al. (1996) JNeurosurg 85:648–654), and many others. Replication-defective retroviralvectors harboring therapeutic polynucleotide sequence as part of theretroviral genome have also been used, particularly with regard tosimple MuLV vectors. See, e.g., Miller et al. (1990) Mol Cell Biol10:4239 (1990); Kolberg (1992) J NIH Res 4:43, and Cornetta et al.(1991) Hum Gene Ther 2:215). Nucleic acid transport coupled toligand-specific, cation-based transport systems (Wu and Wu (1988) J BiolChem, 263:14621–14624) has also been used. Naked DNA expression vectorshave also been described (Nabel et al. (1990), supra); Wolff et al.(1990) Science, 247:1465–1468). In general, these approaches can beadapted to the invention by incorporating nucleic acids encoding theNCSM polypeptides or fragments thereof herein into the appropriatevectors.

General texts which describe gene therapy protocols, which can beadapted to the present invention by introducing the nucleic acids of theinvention into patients, include, e.g., Robbins (1996) Gene TherapyProtocols, Humana Press, NJ, and Joyner (1993) Gene Targeting: APractical Approach, IRL Press, Oxford, England.

Antisense Technology

In addition to expression of the NCSM nucleic acids of the invention asgene replacement nucleic acids, the nucleic acids are also useful forsense and anti-sense suppression of expression, e.g., to down-regulateexpression of a nucleic acid of the invention, once, or when, expressionof the nucleic acid is no-longer desired in the cell. Similarly, thenucleic acids of the invention, or subsequences or anti-sense sequencesthereof, can also be used to block expression of naturally occurringhomologous nucleic acids. A variety of sense and anti-sense technologiesare known in the art, e.g., as set forth in Lichtenstein and Nellen(1997) Antisense Technology: A Practical Approach IRL Press at OxfordUniversity, Oxford, England, and in Agrawal (1996) AntisenseTherapeutics Humana Press, NJ, and the references cited therein.

Use as Probes

Also contemplated are uses of polynucleotides, also referred to hereinas oligonucleotides, typically having at least 12 bases, preferably atleast 15, more preferably at least 20, at least 30, or at least 50 ormore bases, which hybridize under highly stringent conditions to a NCSMpolynucleotide, variant or homologue sequence described herein orfragments thereof. The polynucleotides may be used as probes, primers,sense and antisense agents, and the like, according to methods as notedsupra.

Sequence Variations

Silent Variations

Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide. Forinstance, inspection of the codon table (Table 1) shows that codons AGA,AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, atevery position in a nucleic acid sequence where an arginine is specifiedby a codon, the codon can be altered to any of the corresponding codonsdescribed above without altering the encoded polypeptide. Such nucleicacid variations are “silent variations” are one species of“conservatively modified variations.” It is understood that U in an RNAsequence corresponds to T in a DNA sequence.

TABLE 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It will thus be appreciated by those skilled in the art that due to thedegeneracy of the generic code, a multitude of nucleic acid sequencesencoding NCSM polypeptides of the invention may be produced, some ofwhich may bear minimal sequence homology to the nucleic acid sequencesexplicitly disclosed herein. Using, as an example, the nucleic acidsequence corresponding to nucleotides 1–15 of SEQ ID NO:1, ATG GGT CACACA ATG, a silent variation of this sequence includes ATG GGA CAT ACGATG, both of which sequences encode the amino acid sequence MGHTM, whichcorresponds to amino acids 1–5 of SEQ ID NO:48.

One of ordinary skill in the art will recognize that each codon in anucleic acid (except AUG and UGC, which are ordinarily the only codonfor methionine and tryptophan, respectively) can be modified by standardtechniques to encode a functionally identical polypeptide. Accordingly,each silent variation of a nucleic acid which encodes a polypeptide isimplicit in any described sequence. The invention also provides each andevery possible variation of a nucleic acid sequence encoding a NCSMpolypeptide of the invention that can be made by selecting combinationsbased on possible codon choices. These combinations are made inaccordance with the standard triplet genetic code (codon) (e.g., as setforth in Table 1), as applied to the nucleic acid sequence encoding apolypeptide of the invention or fragment thereof. All such variations ofevery nucleic acid herein are specifically provided and described byconsideration of the sequence in combination with the genetic code. Oneof skill is fully able to generate any silent substitution of thesequences listed herein. For example, the invention includespolynucleotides comprising one or more silent variations of anypolynucleotide sequence selected from SEQ ID NOS:1–21 and 95–142, orcomplementary polynucleotides thereof. Also included are polynucleotidescomprising one or more silent variations of a nucleotide segment orfragment of any polynucleotide sequence selected from SEQ ID NOS:1–21and 95–142, or complementary polynucleotides thereof, wherein suchnucleotide segment or fragment comprises a nucleotide sequence thatencodes a signal peptide and/or extracellular domain of an NCSMpolypeptide or a nucleotide sequence that encodes an extracellulardomain of an NCSM polypeptide. In one such aspect, for example,polynucleotides comprising one or more silent variations of a nucleotidesegment or fragment comprising nucleic acid residues 1–102 (encoding asignal peptide) and/or 103–729 (encoding extracellular domain) of any ofSEQ ID NOS:1–21 and 95–142 are provided. Also provided arepolynucleotides comprising one or more silent variations of anypolynucleotide sequence encoding a polypeptide selected from SEQ IDNOS:48–68, 174,221, 283–295, 290–293, or complementary polynucleotidethereof, and nucleotide segments fragments thereof, including thoseencoding an NCSM signal peptide and/or extracellular domain. Alsoprovided are polypeptides encoded by all such polynucleotides of theinvention comprising one or more silent variations.

Conservative Variations

“Conservatively modified variations,” or simply “conservativevariations,” of a particular nucleic acid sequence refer to thosenucleic acid sequences that encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence of the invention are “conservatively modifiedvariations” where the alterations result in the deletion, addition,and/or substitution of an amino acid with a chemically similar aminoacid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table 2 sets forth six exemplary groupsthat contain amino acids that are “conservative substitutions” for oneanother.

TABLE 2 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Additional groups of amino acids can also be formulated. For example,amino acids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an aliphatic grouping may comprise:Glycine (G), Alanine, Valine, Leucine, Isoleucine. Other groupscontaining amino acids that are conservative substitutions for oneanother include: Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). See also Creighton (1984) Proteins,W. H. Freeman and Company, for additional groupings of amino acids.

Thus, “conservatively substituted variations” of a polypeptide sequenceof the present invention include substitutions of a small percentage,typically less than 5%, more typically less than 4%, 3%, 2%, or 1%, ofthe amino acids of the sequence, with a conservatively selected aminoacid of the same conservative substitution group.

For example, a conservatively substituted variation of the polypeptideidentified herein as SEQ ID NO:48 may contain “conservativesubstitutions,” according to the six groups defined above, in up to 15residues (i.e., 5% of the amino acids) in the 296 amino acidpolypeptide. Listing of a polypeptide or protein sequence herein, inconjunction with the above substitution table, provides an expresslisting of all conservatively substituted polypeptide or proteinsequences.

In a further example, if four conservative substitutions were localizedin the region corresponding to amino acids 69–94 of SEQ ID NO:48,examples of conservatively substituted variations of this region, QKDSKMVLAI LPGKV QVWPE YKNRTI, would include:

NKDSK MVVAI LPGKV QVFPE YKNKTI (SEQ ID NO:294) and

QKDAK MVLAI LPGRV QMWPE YKQRTI (SEQ ID NO:295) and the like, whereconservative substitutions listed in Table 2 (in the above example,conservative substitutions are underlined).

The addition of one or more nucleic acids or sequences that do not alterthe encoded activity of a nucleic acid molecule of the invention, suchas the addition of a non-functional sequence, is a conservativevariation of the basic nucleic acid molecule, and the addition of one ormore amino acid residues that do not alter the activity of a polypeptideof the invention is a conservative variation of the basic polypeptide.Both such types of additions are features of the invention.

One of skill will appreciate that many conservative variations of thenucleic acid sequence constructs that are disclosed yield a functionallyidentical construct. For example, as discussed above, owing to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

In one aspect, the invention includes polynucleotides comprising one ormore conservative variations of any polypeptide selected from SEQ IDNOS:48–68, 174–221, 283–285, and 290–293. Also included are polypeptidescomprising one or more conservative variations of a polypeptide segmentor fragment of any polypeptide sequence selected from SEQ ID NOS:48–68,174–221, 283–285, and 290–293, wherein such polypeptide segment orfragment comprises an amino acid sequence comprising a signal peptideand/or ECD of an NCSM polypeptide or an amino acid sequence comprisingan ECD of an NCSM polypeptide. In one such aspect, polypeptidescomprising at least one conservative variation of a polypeptide segmentor fragment comprising amino acid residues 1–34 (encoding a signalpeptide) and/or 35–243 (encoding an ECD) of any of SEQ ID NOS: 48–68,174–221, 283–285, and 290–293 are provided. Also provided arepolypeptides comprising at least one conservative variation of anypolypeptide sequence encoded by a polynucleotide sequence selected fromSEQ ID NOS:1–21 and 95–142, or complementary polynucleotide thereof, andnucleotide segments fragments thereof, including those encoding an NCSMsignal peptide and/or ECD. Also provided are polynucleotides encoded byall such polynucleotides of the invention comprising one or moreconservative variations.

Nucleic Acid Hybridization

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.)(hereinafter “Tjissen”), as well as in Ausubel, supra, Hames and Higgins(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2, IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other under atleast stringent conditions. The phrase “hybridizing specifically to,”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization wash conditions” and “stringent hybridizationconditions” in the context of nucleic acid hybridization experiments,such as Southern and northern hybridizations, are sequence dependent,and are different under different environmental parameters. An extensiveguide to hybridization of nucleic acids is found in Tijssen (1993),supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

For purposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. (orless) lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH (as noted below, highlystringent conditions can also be referred to in comparative terms). TheT_(m) is the temperature (under defined ionic strength and pH) at which50% of the test sequence hybridizes to a perfectly matched probe. Inother words, the T_(m) indicates the temperature at which the nucleicacid duplex is 50% denatured under the given conditions and itsrepresents a direct measure of the stability of the nucleic acid hybrid.Thus, the T_(m) corresponds to the temperature corresponding to themidpoint in transition from helix to random coil; it depends on length,nucleotide composition, and ionic strength for long stretches ofnucleotides. Typically, under “stringent conditions,” a probe willhybridize to its target subsequence, but to no other sequences. “Verystringent conditions” are selected to be equal to the T_(m) for aparticular probe.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the hybridization temperature) lowers the backgroundsignal, typically with only the specific signal remaining. See, Rapley,R. and Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,Inc. 1998) (hereinafter “Rapley and Walker”), which is incorporatedherein by reference in its entirety for all purposes.

The T_(m) of a DNA-DNA duplex can be estimated using equation (1):T _(m)(° C.)=81.5° C.+16.6(log₁₀ M)+0.41(% G+C)−0.72(% f)−500/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C ) nucleotides, (% f)is the percentage of formalize and n is the number of nucleotide bases(i.e., length) of the hybrid. See, Rapley and Walker, supra.

The T_(m) of an RNA-DNA duplex can be estimated using equation (2):T _(m)(° C.)=79.8° C.+18.5(log₁₀ M)+0.58(% G+C)−11.8(% G+C) ²−0.56(%f)−820/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Id. Equations 1 and 2 above are typicallyaccurate only for hybrid duplexes longer than about 100–200 nucleotides.Id.

The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:T _(m)(° C.)=4(G+C)+2(A+T), where A (adenine), C, T (thymine), and G arethe numbers of the corresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin (orformamide) with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see Sambrook, supra, for adescription of SSC buffer). Often, the high stringency wash is precededby a low stringency wash to remove background probe signal. An examplelow stringency wash is 2×SSC at 40° C. for 15 minutes. An example ofhighly stringent wash conditions is 0.15M NaCl at 72° C. for about 15minutes. An example medium stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4–6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide.

In general, a signal to noise ratio of 2× or 2.5×–5× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization. Detection of atleast stringent hybridization between two sequences in the context ofthe present invention indicates relatively strong structural similarityor homology to, e.g., the nucleic acids of the present inventionprovided in the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringency conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary. See, e.g., Rapley and Walker; Sambrook, all supra.

Comparative hybridization can be used to identify nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. Detection ofhighly stringent hybridization between two nucleotide sequences in thecontext of the present invention indicates relatively strong structuralsimilarity/homology to, e.g., the nucleic acids provided in the sequencelisting herein. Highly stringent hybridization between two nucleotidesequences demonstrates a degree of similarity or homology of structure,nucleotide base composition, arrangement or order that is greater thanthat detected by stringent hybridization conditions. In particular,detection of highly stringent hybridization in the context of thepresent invention indicates strong structural similarity or structuralhomology (e.g., nucleotide structure, base composition, arrangement ororder) to, e.g., the nucleic acids provided in the sequence listingsherein. For example, it is desirable to identify test nucleic acidswhich hybridize to the exemplar nucleic acids herein under stringentconditions.

Thus, one measure of stringent hybridization is the ability to hybridizeto one of the listed nucleic acids of the invention (e.g., nucleic acidsequences SEQ ID NOS:1–47, 95–173, and 253–262, and complementarypolynucleotide sequences thereof) under highly stringent conditions (orvery stringent conditions, or ultra-high stringency hybridizationconditions, or ultra-ultra high stringency hybridization conditions).Stringent hybridization (including, e.g., highly stringent, ultra-highstringency, or ultra-ultra high stringency hybridization conditions) andwash conditions can easily be determined empirically for any testnucleic acid.

For example, in determining highly stringent hybridization and washconditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom SEQ ID NOS:1–47, 95–173, and 253–262, and complementarypolynucleotide sequences thereof, binds to a perfectly matchedcomplementary target (again, a nucleic acid comprising one or morenucleic acid sequences selected from SEQ ID NOS:1–47, 95–173, and253–262, and complementary polynucleotide sequences thereof), with asignal to noise ratio that is at least 2.5×, and optionally 5× or moreas high as that observed for hybridization of the probe to an unmatchedtarget. In this case, the unmatched target is a nucleic acidcorresponding to, e.g., a known B7-1 or related known co-stimulatoryhomologue or the like, e.g., a B7-1 nucleic acid (other than those inthe accompanying sequence listing) present in a public database such asGenBank™ at the time of filing of the subject application. Examples ofsuch unmatched target nucleic acids include, e.g., the following:A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153,AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824,AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833,AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762,AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074,M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593,U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958,Y08823, and Y09950, where the numbers correspond to GenBank accessionnumbers. Additional such sequences can be identified in GenBank by oneof ordinary skill in the art.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 2.5×–10×, typically 5×–10× as high as that observedfor hybridization to any of the unmatched target nucleic acids such as,A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153,AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824,AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833,AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762,AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074,M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593,U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958,Y08823, and Y09950 (where the numbers correspond to GenBank accessionnumbers), or, e.g., other similar known B7-1 or related co-stimulatorysequences or the like presented in GenBank. In one aspect, the inventionprovides a target nucleic acid that, but for the degeneracy of thegenetic code, hybridizes under stringent conditions to a unique codingoligonucleotide that encodes a unique subsequence in a polypeptideselected from SEQ ID NOS:48–94, 174–252, 263–272, and 283–293, where theunique subsequence is unique compared to a polypeptide encoded by any ofabove GenBank Nucleotide Access Nos. For some such nucleic acids, thestringent conditions are selected such that a perfectly complementaryoligonucleotide to the coding oligonucleotide hybridizes to the codingoligonucleotide with at least about a 5× higher signal to noise ratiothan for hybridization of the perfectly complementary oligonucleotide toa control nucleic acid corresponding to any of GenBank NucleotideAccession Nos. set forth above.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids, such as, A92749, A92750, AA983817, AB026121, AB030650,AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896,AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831,AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653,AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533,M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695,S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755,U88622, X60958, Y08823, and Y09950 (where the numbers correspond toGenBank accession numbers), or, e.g., to other similar known B7-1 orco-stimulatory molecule sequences or the like presented in GenBank. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least ½ that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids, such as those represented by: A92749,A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465,AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825,AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834,AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904,AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075,M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925,U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823,and Y09950 (where the numbers correspond to GenBank accession numbers),or, e.g., other similar B7-1 or co-stimulatory sequences or the likepresented in GenBank can be identified. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least ½ that of the perfectly matched complementary targetnucleic acid is said to bind to the probe under ultra-ultra-highstringency conditions.

Target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NOS:1–47, 95–173, and 253–262 under high, ultra-high andultra-ultra high stringency conditions are a feature of the invention.Examples of such nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code, or when antisera generated against one ormore of SEQ ID NOS:48–94, 174–252, 263–272, and 283–293, which has beensubtracted using the polypeptides encoded by known or existing B7-1 orsimilar or related co-stimulatory sequences or the like, including,e.g., those encoded by the following: A92749, A92750, AA983817,AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894,AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829,AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443,AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843,L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191,S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832,U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950 (where thenumbers correspond to GenBank accession numbers), or, e.g., othersimilar B7-1, co-stimulatory sequences, or the like presented in, e.g.,GenBank. Further details on immunological identification of polypeptidesof the invention are found below. Additionally, for distinguishingbetween duplexes with sequences of less than about 100 nucleotides, aTMAC1 hybridization procedure known to those of skill in the art can beused. See, e.g., Sorg, U. et al. 1 Nucleic Acids Res. (Sep. 11, 1991)19(17), incorporated herein by reference in its entirety for allpurposes.

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from any of SEQ IDNOS:1–47, 95–173, and 253–262. The unique subsequence is unique ascompared to a nucleic acid corresponding to any of, e.g., A92749,A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465,AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825,AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834,AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904,AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075,M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925,U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823,and Y09950 (where the numbers correspond to GenBank accession numbers),or, e.g., other similar B7-1 or co-stimulatory sequences or the likepresented in GenBank. Such unique subsequences can be determined byaligning any of SEQ ID NOS:1–47, 95–173, and 253–262 against thecomplete set of nucleic acids, e.g., those corresponding to, e.g.,A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153,AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824,AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833,AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762,AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074,M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593,U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958,Y08823, and Y09950, or other sequences available, e.g., in a publicdatabase, at the filing date of the subject application. Alignment canbe performed using the BLAST algorithm set to default parameters. Anyunique subsequence is useful, e.g., as a probe to identify the nucleicacids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniqueamino acid subsequence in a polypeptide selected from any of SEQ IDNOS:48–94, 174–252, 263–272, and 283–293. Here, the unique subsequenceis unique as compared to a polypeptide or amino acid sequencecorresponding to, e.g., any of A92749, A92750, AA983817, AB026121,AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895,AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830,AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747,AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586,L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541,S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063,U33208, U57755, U88622, X60958, Y08823, and Y09950 (where the numberscorrespond to GenBank accession numbers). Here again, the polypeptide isaligned against the existing polypeptides (the control polypeptides).Note that where the sequence corresponds to a non-translated sequencesuch as a pseudo-gene, the corresponding polypeptide is generated simplyby in silico translation of the nucleic acid sequence into an amino acidsequence, where the reading frame is selected to correspond to thereading frame of homologous NCSM nucleic acids. Such polypeptides areoptionally made by synthetic or recombinant approaches, or can even beordered from companies specializing in polypeptide production.

In addition, the present invention provides a target nucleic acid which,but for codon degeneracy, hybridizes under at least stringent or highlystringent conditions (or conditions of greater stringency) to a uniquecoding oligonucleotide which encodes a unique subsequence in apolypeptide selected from any of SEQ ID NOS:48–94, 174–252, 263–272, and283–293, wherein the unique subsequence is unique as compared to a anamino acid subsequence of a known B7-1 or related co-stimulatorypolypeptide sequence or the like shown in GenBank or to a polypeptidecorresponding to any of the control polypeptides. Unique sequences aredetermined as noted above.

In one example, the stringent conditions are selected such that aperfectly complementary oligonucleotide to the coding oligonucleotidehybridizes to the coding oligonucleotide with at least about a 5–10×higher signal to noise ratio than for hybridization of the perfectlycomplementary oligonucleotide to a control nucleic acid corresponding toany of the control polypeptides. Conditions can be selected such thathigher ratios of signal to noise are observed in the particular assaythat is used, e.g., about 15×, 20×, 30×, 50× or more. In this example,the target nucleic acid hybridizes to the unique coding oligonucleotidewith at least a 2× higher signal to noise ratio as compared tohybridization of the control nucleic acid to the coding oligonucleotide.Again, higher signal to noise ratios can be selected, e.g., about 2.5×,about 5×, about 10×, about 20×, about 30×, about 50× or more. Theparticular signal depends on the label used in the relevant assay, e.g.,a fluorescent label, calorimetric label, radio active label, or thelike.

In another aspect, the invention provides a polypeptide comprising aunique subsequence in a polypeptide selected from any of SEQ IDNOS:48–94, 174–252, 263–272, and 283–293, wherein the unique subsequenceis unique as compared to a polypeptide sequence corresponding to a knownB7-1, co-stimulatory polypeptide or the like, such as, e.g., a B7-1 orco-stimulatory polypeptide sequence present in GenBank.

Percent Sequence Identity—Sequence Similarity

The degree to which one nucleic acid is similar to another provides anindication of whether there is an evolutionary relationship between thetwo or more nucleic acids. In particular, where a high level of sequenceidentity is observed, it is inferred that the nucleic acids are derivedfrom a common ancestor (i.e., that the nucleic acids are homologous). Inaddition, sequence similarity implies similar structural and functionalproperties for the two or more nucleic acids and the sequences theyencode. Accordingly, in the context of the present invention, sequenceswhich have a similar sequence to any given exemplar sequence are afeature of the present invention. In particular, sequences that haveshare percent sequence identities as defined below are a feature of theinvention.

A variety of methods of determining sequence relationships can be used,including manual alignment and computer assisted sequence alignment andanalysis. This later approach is a preferred approach in the presentinvention, due to the increased throughput afforded by computer-assistedmethods. A variety of computer programs for performing sequencealignment are available, or can be produced by one of skill.

As noted above, the sequences of the nucleic acids and polypeptides (andfragments thereof) employed in the subject invention need not beidentical, but can be substantially identical (or substantiallysimilar), to the corresponding sequence of a NCSM polypeptide or nucleicacid molecule (or fragment thereof) or related molecule. For example,the polypeptides can be subject to various changes, such as one or moreamino acid or nucleic acid insertions, deletions, and substitutions,either conservative or non-conservative, including where, e.g., suchchanges might provide for certain advantages in their use, e.g., intheir therapeutic or prophylactic use or administration or diagnosticapplication. The nucleic acids can also be subject to various changes,such as one or more substitutions of one or more nucleic acids in one ormore codons such that a particular codon encodes the same or a differentamino acid, resulting in either a conservative or non-conservativesubstitution, or one or more deletions of one or more nucleic acids inthe sequence. The nucleic acids can also be modified to include one ormore codons that provide for optimum expression in an expression system(e.g., mammalian cell or mammalian expression system), while, ifdesired, said one or more codons still encode the same amino acid(s).Such nucleic acid changes might provide for certain advantages in theirtherapeutic or prophylactic use or administration, or diagnosticapplication. The nucleic acids and polypeptides can be modified in anumber of ways so long as they comprise a sequence substantiallyidentical (as defined below) to a sequence in a respective NCSM nucleicacid or polypeptide molecule.

Alignment and comparison of relatively short amino acid sequences (lessthan about 30 residues) is typically straightforward. Comparison oflonger sequences can require more sophisticated methods to achieveoptimal alignment of two sequences. Optimal alignment of sequences foraligning a comparison window can be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv Appl Math 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol48:443, by the search for similarity method of Pearson and Lipman (1988)Proc Natl Acad Sci USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.; and BLAST, see, e.g., Altschul et al. (1977) Nuc AcidsRes 25:3389–3402 and Altschul et al. (1990) J Mol Biol 215:403–410), orby inspection, with the best alignment (i.e., resulting in the highestpercentage of sequence similarity or sequence identity over thecomparison window) generated by the various methods being selected.

The term “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The term “sequence identity” or “percent identity” (“% identity”) meansthat two polynucleotide or polypeptide sequences are identical (i.e., ona nucleotide-by-nucleotide basis or amino acid-by-amino acid basis,respectively) over a window of comparison. The term “percentage ofsequence identity” (or “percent sequence identity” or simply “percentidentity” or “% identity”) or “percentage of sequence similarity” (or“percent sequence similarity” or simply “percent similarity”) iscalculated by comparing two optimally aligned polynucleotide orpolypeptide sequences over the window of comparison, determining thenumber of positions at which the identical residues occur in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity (or percentage of sequencesimilarity). Thus, for example, with regard to polypeptide sequences,the term sequence identity means that two polypeptide sequences areidentical (on an amino acid-by-amino acid basis) over a window ofcomparison, and a percentage of amino acid residue sequence identity (orpercentage of amino acid residue sequence similarity), can becalculated. For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Maximum correspondence can be determinedby using one of the sequence algorithms described herein (or otheralgorithms available to those of ordinary skill in the art) or by visualinspection.

The phrase “substantially identical” or “substantial identity” in thecontext of two nucleic acids or polypeptides, refers to two or moresequences or subsequences that have at least about 50%, 60%, 70%, 75%,preferably 80% or 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, or more nucleotide or amino acid residue %identity, respectively, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. In certain embodiments,the substantial identity exists over a region of amino acid sequencesthat is at least about 50 residues in length, preferably over a regionof at least about 100 residues in length, and more preferably thesequences are substantially identical over at least about 150, 200, or250 amino acid residues. In certain aspects, substantial identity existsover a region of nucleic acid sequences of at least about 500 residues,preferably over a region of at least about 600 residues in length, andmore preferably the sequences are substantially identical over at leastabout 700, 800, or 850 nucleic acid residues. In some aspects, the aminoacid or nucleic acid sequences are substantially identical over theentire length of the corresponding coding region.

As applied to polypeptides and peptides, the term “substantial identity”typically means that two polypeptide or peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights (described in detail below) or by visual inspection, shareat least about 60% or 70%, often at least 75%, preferably at least about80% or 85%, more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% or more percent amino acid residue sequenceidentity or sequence similarity. Similarly, as applied in the context oftwo nucleic acids, the term substantial identity or substantialsimilarity means that the two nucleic acid sequences, when optimallyaligned, such as by the programs BLAST, GAP or BESTFIT using default gapweights (described in detail below) or by visual inspection, share atleast about 60 percent, 70 percent, or 80 percent sequence identity orsequence similarity, preferably at least about 90 percent amino acidresidue sequence identity or sequence similarity, more preferably atleast about 95 percent sequence identity or sequence similarity, or more(including, e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99,99.5, or more percent nucleotide sequence identity or sequencesimilarity).

In one aspect, the present invention provides nucleic acids encodingNCSM amino acid molecules (e.g., full-length polypeptide, signalpeptide, ECD, cytoplasmic domain, transmembrane domain, mature region,or other fragment) having at least about 60%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or morepercent sequence identity or sequence similarity with the nucleic acidof any of SEQ ID NOS:1–47, 95–173, and 253–262 or a fragment thereof,including, e.g., one or more of a signal peptide, ECD, cytoplasmicdomain, transmembrane domain, or mature region or any combinationthereof. Some such encoded polypeptides have the CD28BP or CTLA-4BPproperties described herein.

In another aspect, the present invention provides NCSM polypeptides(e.g., full-length NCSM polypetide, signal peptide, ECD, cytoplasmicdomain, transmembrane domain, mature region, or other fragment), andfusion proteins comprising said polypeptides, having at least about 50%,60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5% or more percent sequence identity or sequence similarity withthe polypeptide of any of SEQ ID NOS:48–94, 174–252, 263–272, and283–293 or a fragment thereof, including, e.g., one or more of a signalpeptide, ECD, cytoplasmic domain, transmembrane domain, or mature regionor any combination thereof. Such fragments of SEQ ID NOS:69–92, 222–272,and 286–288 may have at least one CTLA-4BP property described herein,such as, e.g., an ability to inhibit T cell proliferation or activationin conjunction with stimulation of T cell receptor (e.g., by antigen oranti-CD3 Ab) and/or a CTLA-4/CD28 binding affinity ratio about equal toor greater than that of hB7-1. Such fragments of SEQ ID NOS:48–68,174–221, 283–285, and 289–293 may have at least one CD28BP propertydescribed herein, such as, e.g., an ability to induce T cellproliferation or activation in conjunction with stimulation of T cellreceptor (e.g., by antigen or anti-CD3 Ab) and/or a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than that of hB7-1. Suchfragments of SEQ ID NOS:93–94 may have an ability to induce T cellproliferation or activation in conjunction with stimulation of T cellreceptor (by, e.g., an antigen) and/or a CD28/CTLA-4 binding affinityratio approximately equal to that of a primate, such as hB7-1.

In yet another aspect, the present invention provides NCSM homologuepolypeptides that are substantially identical or substantially similarover at least about 150, 180, 170, 190, 200, 210, 225, 230, 240, 250,275, or 285 or more contiguous amino acids of at least one of SEQ IDNOS:69–92, 222–272, and 286–288; some such polypeptides may have anability to inhibit T cell proliferation or activation and/or aCTLA-4/CD28 binding affinity ratio about equal to or greater than thatof hB7-1 as described herein.

In yet another aspect, the present invention provides NCSM homologuepolypeptides that are substantially identical or substantially similarover at least about 150, 180, 170, 190, 200, 210, 225, 230, 240, 250,275, or 285 or more contiguous amino acids of at least one of SEQ IDNOS:48–68, 174–221, 283–285, and 289–293; some such polypeptides mayhave an ability to induce T cell proliferation or activation inconjunction with stimulation of T cell receptor (e.g., by antigen oranti-CD3 Ab) and/or a CD28/CTLA-4 binding affinity ratio about equal toor greater than that of hB7-1.

NCSM homologue polypeptides that are substantially identical orsubstantially similar over at least about 150, 180, 170, 190, 200, 210,225, 230, 240, 250, 275, or 285 or more contiguous amino acids of atleast one of SEQ ID NOS:93–94; some such polypeptides may have anability to induce T cell proliferation or activation in conjunction withstimulation of T cell receptor (by, e.g., anti-CD3 Ab or antigen) and/ora CD28/CTLA-4 binding affinity ratio about equal to that of a primate,such as hB7-1.

A feature of the invention is a NCSM polypeptide comprising at least 175contiguous amino acids of any one of SEQ ID NOS:48–94, 174–252, 263–272,and 283–293. In other embodiments, the polypeptide comprises about 175,200, 210, 225, 275, or more contiguous amino acid residues of any one ofSEQ ID NOS:48–94, 174–252, 263–272, and 283–293. In other embodiments,the polypeptide is at least about 280 amino acids, and still morepreferably at least about 285 amino acids in length.

Alternatively, parameters are set such that one or more sequences of theinvention are identified by alignment to a query sequence selected fromamong SEQ ID NOS:49–94, 174–252, 263–272 and 283–293, while sequencescorresponding to unrelated polypeptides, e.g., those encoded by knownnucleic acid sequences represented by GenBank accession numbers (e.g.,known B7-1 sequences) are not identified.

Preferably, residue positions that are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitution refers to the interchange-ability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, arginine-lysine-histidine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Alignment and comparison of relatively short amino acid sequences (lessthan about 30 residues) is typically straightforward. Comparison oflonger sequences can require more sophisticated methods to achieveoptimal alignment of two sequences. Optimal alignment of sequences foraligning a comparison window can be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv Appl Math 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol48:443, by the search for similarity method of Pearson and Lipman (1988)Proc Natl Acad Sci USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection, with the best alignment (i.e.,resulting in the highest percentage of sequence similarity over thecomparison window) generated by the various methods being selected.

A preferred example of an algorithm that is suitable for determiningpercent sequence identity (percent identity) and sequence similarity isthe FASTA algorithm, which is described in Pearson, W. R. & Lipman, D.J. (1988) Proc Natl Acad Sci USA 85:2444. See also, W. R. Pearson (1996)Methods Enzymology 266:227–258. Preferred parameters used in a FASTAalignment of DNA sequences to calculate percent identity are optimized,BL50 Matrix 15: −5, k-tuple=2; joining penalty=40, optimization=28; gappenalty −12, gap length penalty=−2; and width=16.

Other preferred examples of algorithm that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1997) NucAcids Res 25:3389–3402 and Altschul et al. (1990) J Mol Biol215:403–410, respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program (e.g., BLASTP 2.0.14; Jun. 29,2000) uses as defaults a wordlength of 3, and expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc NatlAcad Sci USA 89:10915) uses alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. Again, as with othersuitable algorithms, the stringency of comparison can be increased untilthe program identifies only sequences that are more closely related tothose in the sequence listings herein (i.e., SEQ ID NOS:1–47, 95–173,and 253–262 or, alternatively, SEQ ID NOS:48–94, 174–252, 263–272, and283–293, rather than sequences that are more closely related to othersimilar sequences such as, e.g., those nucleic acid sequencesrepresented by GenBank accession numbers set forth herein, and or othersimilar molecules found in, e.g., GenBank. In other words, thestringency of comparison of the algorithms can be increased so that allknown prior art (e.g., those represented by GenBank accession numbersshown herein, or other similar molecules found in, e.g., GenBank) isexcluded.

The BLAST algorithm also performs a statistical analysis of thesimilarity or identity between two sequences (see, e.g., Karlin &Altschul (1993) Proc Natl Acad Sci USA 90:5873–5787). One measure ofsimilarity provided by this algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencesequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity or percent sequence similarity. It also plots a treeor dendogram showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J Mol Evol 35:351–360. The method usedis similar to the method described by Higgins & Sharp (1989) CABIOS5:151–153. The program can align up to 300 sequences, each of a maximumlength of 5,000 nucleotides or amino acids. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This cluster isthen aligned to the next most related sequence or cluster of alignedsequences. Two clusters of sequences are aligned by a simple extensionof the pairwise alignment of two individual sequences. The finalalignment is achieved by a series of progressive, pairwise alignments.The program is run by designating specific sequences and their aminoacid or nucleotide coordinates for regions of sequence comparison and bydesignating the program parameters. Using PILEUP, a reference sequenceis compared to other test sequences to determine the percent sequenceidentity (or percent sequence similarity) relationship using thefollowing parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal. (1984) Nuc Acids Res 12:387–395).

Another preferred example of an algorithm that is suitable for multipleDNA and amino acid sequence alignments is the CLUSTALW program(Thompson, J. D. et al. (1994) Nuc Acids Res 22:4673–4680). CLUSTALWperforms multiple pairwise comparisons between groups of sequences andassembles them into a multiple alignment based on homology. Gap open andGap extension penalties were 10 and 0.05 respectively. For amino acidalignments, the BLOSUM algorithm can be used as a protein weight matrix(Henikoff and Henikoff (1992) Proc Natl Acad Sci USA 89:10915–10919).Another example of an algorithm suitable for multiple DNA and amino acidsequence alignments is the Jotun Hein method, Hein (1990), from withinthe MegaLine™ DNASTAR package (MegaLine™ Version 4.03, manufactured byDNASTAR, Inc.) used according to the manufacturer's instructions anddefault values specified in the program.

It will be understood by one of ordinary skill in the art, that theabove discussion of search and alignment algorithms also applies toidentification and evaluation of polynucleotide sequences, with thesubstitution of query sequences comprising nucleotide sequences, andwhere appropriate, selection of nucleic acid databases.

Substrates and Formates for Sequence Recombination

The polynucleotides of the invention and fragments thereof areoptionally used as substrates for any of a variety of recombination andrecursive sequence recombination reactions, in addition to their use instandard cloning methods as set forth in, e.g., Ausubel, Berger andSambrook, e.g., to produce additional NCSM polynucleotides or fragmentsthereof that encode polypeptides and fragments thereof having withdesired properties. A variety of such reactions are known, includingthose developed by the inventors and their co-workers.

DNA recombination is a method for generating and identifying new NCSMmolecules, e.g., including those with altered relative bindingcapacities to either or both of the CD28 and CTLA-4 receptors (ascompared to, e.g., hB7-1) and altered functional activities, including,e.g., altered capacities to induce or inhibit T cell activation and/ordifferentiation, induce or inhibit cytokine production, and/or promoteor inhibit anergy and/or tolerance as described herein.

A variety of diversity generating protocols for generating andidentifying NCSM molecules having one of more of the propertiesdescribed herein are available and described in the art. The procedurescan be used separately, and/or in combination to produce one or moreNCSM variants of a nucleic acid or set of nucleic acids, as wellvariants of encoded proteins. Individually and collectively, theseprocedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics. While distinctions andclassifications are made in the course of the ensuing discussion forclarity, it will be appreciated that the techniques are often notmutually exclusive. Indeed, the various methods can be used singly or incombination, in parallel or in series, to access diverse sequencevariants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties, or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein, or otherwise available to one of skill, any nucleic acids thatare generated or produced can be selected for a desired activity orproperty, e.g. ability to induce or inhibit T cell proliferation oractivation, cytokine production, alter binding affinity to one or moreof CD28 or CTLA-4 receptors. This can include identifying any activitythat can be detected, for example, in an automated or automatableformat, by any of the assays in the art and/or the assays of theinvention discussed here and/or in the Example section below. A varietyof related (or even unrelated) properties can be evaluated, in serial orin parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences that encode NCSM polypeptidesas described herein are found in the following publications and thereferences cited therein: Soong, N. et al. (2000) “Molecular breeding ofviruses” Nat Genet 25(4):436–439; Stemmer, et al. (1999) “Molecularbreeding of viruses for targeting and other clinical properties” TumorTargeting 4:1–4; Ness et al. (1999) “DNA Shuffling of subgenomicsequences of subtilisin” Nature Biotechnology 17:893–896; Chang et al.(1999) “Evolution of a cytokine using DNA family shuffling” NatureBiotechnology 17:793–797; Minshull and Stemmer (1999) “Protein evolutionby molecular breeding” Current Opinion in Chemical Biology 3:284–290;Christians et al. (1999) “Directed evolution of thymidine kinase for AZTphosphorylation using DNA family shuffling” Nature Biotechnology17:259–264; Crameri et al. (1998) “DNA shuffling of a family of genesfrom diverse species accelerates directed evolution” Nature 391:288–291;Crameri et al. (1997) “Molecular evolution of an arsenate detoxificationpathway by DNA shuffling,” Nature Biotechnology 15:436–438; Zhang et al.(1997) “Directed evolution of an effective fucosidase from agalactosidase by DNA shuffling and screening” Proc. Natl. Acad. Sci. USA94:4504–4509; Patten et al. (1997) “Applications of DNA Shuffling toPharmaceuticals and Vaccines” Current Opinion in Biotechnology8:724–733; Crameri et al. (1996) “Construction and evolution ofantibody-phage libraries by DNA shuffling” Nature Medicine 2:100–103;Crameri et al. (1996) “Improved green fluorescent protein by molecularevolution using DNA shuffling” Nature Biotechnology 14:315–319; Gates etal. (1996) “Affinity selective isolation of ligands from peptidelibraries through display on a lac repressor ‘headpiece dimer’” Journalof Molecular Biology 255:373–386; Stemmer (1996) “Sexual PCR andAssembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers,New York. pp. 447–457; Crameri and Stemmer (1995) “Combinatorialmultiple cassette mutagenesis creates all the permutations of mutant andwildtype cassettes” BioTechniques 18:194–195; Stemmer et al., (1995)“Single-step assembly of a gene and entire plasmid form large numbers ofoligodeoxy-ribonucleotides” Gene, 164:49–53; Stemmer (1995) “TheEvolution of Molecular Computation” Science 270: 1510; Stemmer (1995)“Searching Sequence Space” Bio/Technology 13:549–553; Stemmer (1994)“Rapid evolution of a protein in vitro by DNA shuffling” Nature370:389–391; and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution.” Proc.Natl. Acad. Sci. USA 91:10747–10751.

The term “shuffling” is used herein to indicate recombination betweennon-identical sequences, in some embodiments shuffling may includecrossover via homologous recombination or via non-homologousrecombination, such as via cre/lox and/or flp/frt systems. Shuffling canbe carried out by employing a variety of different formats, includingfor example, in vitro and in vivo shuffling formats, in silico shufflingformats, shuffling formats that utilize either double-stranded orsingle-stranded templates, primer based shuffling formats, nucleic acidfragmentation-based shuffling formats, and oligonucleotide-mediatedshuffling formats, all of which are based on recombination eventsbetween non-identical sequences and are described in more detail orreferenced herein below, as well as other similar recombination-basedformats.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157–178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369–374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423–462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193–1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1–7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488–492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367–382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240–245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468–500(1983); Methods in Enzymol. 154: 329–350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487–6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468–500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329–350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749–8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765–8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679–9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791–802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803–814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441–9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350–367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987–6999).

Additional suitable methods include point mismatch repair (Kramer et al.(1984) “Point Mismatch Repair” Cell 38:879–887), mutagenesis usingrepair-deficient host strains (Carter et al. (1985) “Improvedoligonucleotide site-directed mutagenesis using M13 vectors” Nucl. AcidsRes. 13: 4431–4443; and Carter (1987) “Improved oligonucleotide-directedmutagenesis using M13 vectors” Methods in Enzymol. 154: 382–403),deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) “Use ofoligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-purification (Wells et al.(1986) “Importance of hydrogen-bond formation in stabilizing thetransition state of subtilisin” Phil. Trans. R. Soc. Lond. A 317:415–423), mutagenesis by total gene synthesis (Nambiar et al. (1984)“Total synthesis and cloning of a gene coding for the ribonuclease Sprotein” Science 223: 1299–1301; Sakamar and Khorana (1988) “Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin)” Nucl. AcidsRes. 14: 6361–6372; Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315–323; and Grundström et al. (1985) “Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res.13: 3305–3316), double-strand break repair (Mandecki (1986)“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177–7181; and Arnold (1993) “Protein engineering forunusual environments” Current Opinion in Biotechnology 4:450–455).Additional details on many of the above methods can be found in Methodsin Enzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following U.S. patents, PCT publications and applications,and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,1997), “Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 toStemmer et al. (Sep. 22, 1998) “Methods for Generating Polynucleotideshaving Desired Characteristics by Iterative Selection andRecombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S.Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-ComplementaryPolymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov.17, 1998), “Methods and Compositions for Cellular and MetabolicEngineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by RandomFragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “EndComplementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer andCrameri “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” WO 97/35966by Minshull and Stemmer, “Methods and Compositions for Cellular andMetabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting ofGenetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “AntigenLibrary Immunization;” WO 99/41369 by Punnonen et al. “Genetic VaccineVector Engineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination;” WO 00/18906 by Patten etal., “Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre etal. “Evolution of Whole Cells and Organisms by Recursive Recombination;”WO 00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 00/42559 by Selifonov and Stemmer “Methods ofPopulating Data Structures for Use in Evolutionary Simulations;” WO00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides & Polypeptides Having Desired Characteristics;”PCT/US00/26708 by Welch et al., “Use of Codon-Varied OligonucleotideSynthesis for Synthetic Shuffling;” and PCT/US01/06775 “Single-StrandedNucleic Acid Template-Mediated Recombination and Nucleic Acid FragmentIsolation” by Affholter.

Several different general classes of sequence modification methods, suchas mutation, recombination, etc. are applicable to the present inventionand set forth, e.g., in the references above and below. That is, nucleicacids encoding CD28BP or CTLA-4BP polypeptides can be diversified by anyof the methods described herein, e.g., including various mutation andrecombination methods, individually or in combination, to generatenucleic acids with a desired activity or property, including, e.g.,those described herein, such as an ability to enhance an immuneresponse, such as by inducing T cell activation or proliferation, anability to down-regulate or inhibit an immune response, such as byinhibiting T cell activation or proliferation, an ability topreferentially bind and/or signal through either or both CD28 and CTLA-4receptors.

The following exemplify some of the different types of preferred formatsfor diversity generation in the context of the present invention,including, e.g., certain recombination based diversity generationformats.

Nucleic acids can be recombined in vitro by any of a variety oftechniques discussed in the references above, including e.g., DNAsedigestion of nucleic acids to be recombined followed by ligation and/orPCR reassembly of the nucleic acids. For example, sexual PCR mutagenesiscan be used in which random (or pseudo random, or even non-random)fragmentation of the DNA molecule is followed by recombination, based onsequence similarity, between DNA molecules with different but relatedDNA sequences, in vitro, followed by fixation of the crossover byextension in a polymerase chain reaction. This process and many processvariants is described in several of the references above, e.g., inStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747–10751. Thus, nucleicacids encoding B7-1 polypeptides and subsequences thereof can berecombined in vitro to generate nucleic acids encoding modified orrecombinant B7-1 polypeptides, NCSM polypeptides, CD28BP polypeptides,CTLA-4BP polypeptides, or subsequences thereof, each of which has one ormore desired properties, including those described herein.

Similarly, nucleic acids can be recursively recombined in vivo, e.g., byallowing recombination to occur between nucleic acids in cells. Manysuch in vivo recombination formats are set forth in the references notedabove. Such formats optionally provide direct recombination betweennucleic acids of interest, or provide recombination between vectors,viruses, plasmids, etc., comprising the nucleic acids of interest, aswell as other formats. Details regarding such procedures are found inthe references noted above. Thus, nucleic acids encoding B7-1polypeptides and subsequences thereof can be recombined in vivo priorto, or in concert with screening and/or selection procedures to identifymodified or recombinant B7-1 polypeptides, NCSM polypeptides, CD28BPpolypeptides, CTLA-4BP polypeptides, or subsequences thereof, each ofwhich has one or more desired properties, including those describedherein. Whole genome recombination methods can also be used in whichwhole genomes of cells or other organisms are recombined, optionallyincluding spiking of the genomic recombination mixtures with desiredlibrary components (e.g., genes corresponding to the pathways of thepresent invention). These methods have many applications, includingthose in which the identity of a target gene is not known. Details onsuch methods are found, e.g., in WO 98/31837 by del Cardayre et al.“Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” and in, e.g., PCT/US99/15972 by del Cardayre et al.,also entitled “Evolution of Whole Cells and Organisms by RecursiveSequence Recombination.”

Synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest (e.g., B7polypeptides) are synthesized and reassembled in PCR or ligationreactions which include oligonucleotides which correspond to more thanone parental nucleic acid, thereby generating new recombined nucleicacids. Oligonucleotides can be made by standard nucleotide additionmethods, or can be made, e.g., by tri-nucleotide synthetic approaches.Details regarding such approaches are found in the references notedabove, including, e.g., WO 00/42561 by Crameri et al., “OligonucleotideMediated Nucleic Acid Recombination;” PCT/US00/26708 by Welch et al.,“Use of Codon-Varied Oligonucleotide Synthesis for Synthetic Shuffling;”WO 00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides and Polypeptides Having Desired Characteristics;” and WO00/42559 by Selifonov and Stemmer “Methods of Populating Data Structuresfor Use in Evolutionary Simulations.”

In silico methods of recombination can be effected in which geneticalgorithms are used in a computer to recombine sequence strings whichcorrespond to homologous (or even non-homologous) nucleic acids. Theresulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotidesynthesis/gene reassembly techniques. This approach can generate random,partially random or designed variants. Many details regarding in silicorecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 by Selifonov et al.,“Methods for Making Character Strings, Polynucleotides and PolypeptidesHaving Desired Characteristics” and WO 00/42559 by Selifonov and Stemmer“Methods of Populating Data Structures for Use in EvolutionarySimulations.” Extensive details regarding in silico recombinationmethods are found in these applications. This methodology is generallyapplicable to the present invention in providing for recombination ofthe character strings corresponding to nucleic acids encodingco-stimulatory B7 molecules in silico and/or the generation ofcorresponding nucleic acids or proteins.

Many methods of accessing natural diversity, e.g., by hybridization ofdiverse nucleic acids or nucleic acid fragments to single-strandedtemplates, followed by polymerization and/or ligation to regeneratefull-length sequences, optionally followed by degradation of thetemplates and recovery of the resulting modified nucleic acids can besimilarly used. In one method employing a single-stranded template, thefragment population derived from the genomic library(ies) is annealedwith partial, or, often approximately full length ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in “Single-Stranded Nucleic AcidTemplate-Mediated Recombination and Nucleic Acid Fragment Isolation” byAffholter, PCT/US01/06775.

In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein.

Any of the preceding general recombination formats can be practiced in areiterative fashion (e.g., one or more cycles of mutation/recombinationor other diversity generation methods, optionally followed by one ormore selection methods) to generate a more diverse set of recombinantnucleic acids.

Mutagenesis employing polynucleotide chain termination methods have alsobeen proposed (see e.g., U.S. Pat. No. 5,965,408, “Method of DNAreassembly by interrupting synthesis” to Short, and the referencesabove), and can be applied to the present invention. In this approach,double stranded DNAs corresponding to one or more genes sharing regionsof sequence similarity are combined and denatured, in the presence orabsence of primers specific for the gene. The single strandedpolynucleotides are then annealed and incubated in the presence of apolymerase and a chain terminating reagent (e.g., ultraviolet, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., containing partially extended chains, are thendenatured and reannealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are diversified with respect to thestarting population of DNA molecules. Optionally, the products, orpartial pools of the products, can be amplified at one or more stages inthe process. Polynucleotides produced by a chain termination method,such as described above, are suitable substrates for any other describedrecombination format.

Diversity also can be generated in nucleic acids or populations ofnucleic acids using a recombinational procedure termed “incrementaltruncation for the creation of hybrid enzymes” (“ITCHY”) described inOstermeier et al. (1999) “A combinatorial approach to hybrid enzymesindependent of DNA homology” Nature Biotech 17:1205. This approach canbe used to generate an initial a library of variants which canoptionally serve as a substrate for one or more in vitro or in vivorecombination methods. See, also, Ostermeier et al. (1999)“Combinatorial Protein Engineering by Incremental Truncation,” Proc.Natl. Acad. Sci. USA, 96: 3562–67; Ostermeier et al. (1999),“Incremental Truncation as a Strategy in the Engineering of NovelBiocatalysts,” Biological and Medicinal Chemistry, 7: 2139–44.

Mutational methods which result in the alteration of individualnucleotides or groups of contiguous or non-contiguous nucleotides can befavorably employed to introduce nucleotide diversity. For example,mutagenesis procedures resulting in changes of one or more nucleotidescan be used to generate any number of nucleic acids encodingpolypeptides of the present invention. Many mutagenesis methods arefound in the above-cited references; additional details regardingmutagenesis methods can be found in following, which can also be appliedto the present invention.

For example, error-prone PCR can be used to generate nucleic acidvariants. Using this technique, PCR is performed under conditions wherethe copying fidelity of the DNA polymerase is low, such that a high rateof point mutations is obtained along the entire length of the PCRproduct. Examples of such techniques are found in the references aboveand, e.g., in Leung et al. (1989) Technique 1:11–15 and Caldwell et al.(1992) PCR Methods Applic. 2:28–33. Similarly, assembly PCR can be used,in a process which involves the assembly of a PCR product from a mixtureof small DNA fragments. A large number of different PCR reactions canoccur in parallel in the same reaction mixture, with the products of onereaction priming the products of another reaction.

Oligonucleotide directed mutagenesis can be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science, 241:53–57. Similarly, cassettemutagenesis can be used in a process that replaces a small region of adouble stranded DNA molecule with a synthetic oligonucleotide cassettethat differs from the native sequence. The oligonucleotide can contain,e.g., completely and/or partially randomized native sequence(s).

Recursive ensemble mutagenesis is a process in which an algorithm forprotein mutagenesis is used to produce diverse populations ofphenotypically related mutants, members of which differ in amino acidsequence. This method uses a feedback mechanism to monitor successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811–7815.

Exponential ensemble mutagenesis can be used for generatingcombinatorial libraries with a high percentage of unique and functionalmutants. Small groups of residues in a sequence of interest arerandomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures arein Delegrave & Youvan (1993) Biotechnology Research 11: 1548–1552.

In vivo mutagenesis can be used to generate random mutations in anycloned DNA of interest by propagating the DNA, e.g., in a strain of E.coli that carries mutations in one or more of the DNA repair pathways.These “mutator” strains have a higher random mutation rate than that ofa wild-type parent. Propagating the DNA in one of these strains willeventually generate random mutations within the DNA. Such procedures aredescribed in the references noted above.

Other procedures for introducing diversity into a genome, e.g. abacterial, fungal, animal or plant genome can be used in conjunctionwith the above described and/or referenced methods. For example, inaddition to the methods above, techniques have been proposed whichproduce nucleic acid multimers suitable for transformation into avariety of species (see, e.g., Schellenberger U.S. Pat. No. 5,756,316and the references above). Transformation of a suitable host with suchmultimers, consisting of genes that are divergent with respect to oneanother, (e.g., derived from natural diversity or through application ofsite directed mutagenesis, error prone PCR, passage through mutagenicbacterial strains, and the like), provides a source of nucleic aciddiversity for DNA diversification, e.g., by an in vivo recombinationprocess as indicated above.

Alternatively, a multiplicity of monomeric polynucleotides sharingregions of partial sequence similarity can be transformed into a hostspecies and recombined in vivo by the host cell. Subsequent rounds ofcell division can be used to generate libraries, members of which,include a single, homogenous population, or pool of monomericpolynucleotides. Alternatively, the monomeric nucleic acid can berecovered by standard techniques, e.g., PCR and/or cloning, andrecombined in any of the recombination formats, including recursiverecombination formats, described above.

Methods for generating multispecies expression libraries have beendescribed (in addition to the reference noted above, see, e.g., Petersonet al. (1998) U.S. Pat. No. 5,783,431 “METHODS FOR GENERATING ANDSCREENING NOVEL METABOLIC PATHWAYS,” and Thompson, et al. (1998) U.S.Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVEL METABOLICPATHWAYS) and their use to identify protein activities of interest hasbeen proposed (In addition to the references noted above, see Short(1999) U.S. Pat. No. 5,958,672 “PROTEIN ACTIVITY SCREENING OF CLONESHAVING DNA FROM UNCULTIVATED MICROORGANISMS”). Multispecies expressionlibraries include, in general, libraries comprising cDNA or genomicsequences from a plurality of species or strains, operably linked toappropriate regulatory sequences, in an expression cassette. The cDNAand/or genomic sequences are optionally randomly ligated to furtherenhance diversity. The vector can be a shuttle vector suitable fortransformation and expression in more than one species of host organism,e.g., bacterial species, eukaryotic cells. In some cases, the library isbiased by preselecting sequences which encode a protein of interest, orwhich hybridize to a nucleic acid of interest. Any such libraries can beprovided as substrates for any of the methods herein described.

The above-described procedures have been largely directed to increasingnucleic acid and/or encoded protein diversity. However, in many cases,not all of the diversity is useful, e.g., functional, and contributesmerely to increasing the background of variants that must be screened orselected to identify the few favorable variants. In some applications,it is desirable to preselect or prescreen libraries (e.g., an amplifiedlibrary, a genomic library, a cDNA library, a normalized library, etc.)or other substrate nucleic acids prior to diversification, e.g., byrecombination-based mutagenesis procedures, or to otherwise bias thesubstrates towards nucleic acids that encode functional products. Forexample, in the case of antibody engineering, it is possible to bias thediversity generating process toward antibodies with functional antigenbinding sites by taking advantage of in vivo recombination events priorto manipulation by any of the described methods. For example, recombinedCDRs derived from B cell cDNA libraries can be amplified and assembledinto framework regions (e.g., Jirholt et al. (1998) “Exploiting sequencespace: shuffling in vivo formed complementarity determining regions intoa master framework” Gene 215: 471) prior to diversifying according toany of the methods described herein.

Libraries can be biased towards nucleic acids which encode proteins withdesirable enzyme activities. For example, after identifying a clone froma library which exhibits a specified activity, the clone can bemutagenized using any known method for introducing DNA alterations. Alibrary comprising the mutagenized homologues is then screened for adesired activity, which can be the same as or different from theinitially specified activity. An example of such a procedure is proposedin Short (1999) U.S. Pat. No. 5,939,250 for “PRODUCTION OF ENZYMESHAVING DESIRED ACTIVITIES BY MUTAGENESIS.” Desired activities can beidentified by any method known in the art. For example, WO 99/10539proposes that gene libraries can be screened by combining extracts fromthe gene library with components obtained from metabolically rich cellsand identifying combinations which exhibit the desired activity. It hasalso been proposed (e.g., WO 98/58085) that clones with desiredactivities can be identified by inserting bioactive substrates intosamples of the library, and detecting bioactive fluorescencecorresponding to the product of a desired NCSM activity as describedherein using a fluorescent analyzer, e.g., a flow cytometry device, aCCD, a fluorometer, or a spectrophotometer.

Libraries can also be biased towards nucleic acids which have specifiedcharacteristics, e.g., hybridization to a selected nucleic acid probe.For example, application WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences in the following manner.Single stranded DNA molecules from a population of genomic DNA arehybridized to a ligand-conjugated probe. The genomic DNA can be derivedfrom either a cultivated or uncultivated microorganism, or from anenvironmental sample. Alternatively, the genomic DNA can be derived froma multicellular organism, or a tissue derived therefrom. Second strandsynthesis can be conducted directly from the hybridization probe used inthe capture, with or without prior release from the capture medium or bya wide variety of other strategies known in the art. Alternatively, theisolated single-stranded genomic DNA population can be fragmentedwithout further cloning and used directly in, e.g., arecombination-based approach, that employs a single-stranded template,as described above.

“Non-Stochastic” methods of generating nucleic acids and polypeptidesare alleged in Short “Non-Stochastic Generation of Genetic Vaccines andEnzymes” WO 00/46344. These methods, including proposed non-stochasticpolynucleotide reassembly and site-saturation mutagenesis methods beapplied to the present invention as well. Random or semi-randommutagenesis using doped or degenerate oligonucleotides is also describedin, e.g., Arkin and Youvan (1992) “Optimizing nucleotide mixtures toencode specific subsets of amino acids for semi-random mutagenesis”Biotechnology 10:297–300; Reidhaar-Olson et al. (1991) “Randommutagenesis of protein sequences using oligonucleotide cassettes”Methods Enzymol. 208:564–86; Lim and Sauer (1991) “The role of internalpacking interactions in determining the structure and stability of aprotein” J. Mol. Biol. 219:359–76; Breyer and Sauer (1989) “Mutationalanalysis of the fine specificity of binding of monoclonal antibody 51Fto lambda repressor” J. Biol. Chem. 264:13355–60); and “Walk-ThroughMutagenesis” (Crea, R; U.S. Pat. Nos. 5,830,650 and 5,798,208, and EPPatent 0527809 B1.

It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods.

Kits for mutagenesis, library construction and other diversitygeneration methods are also commercially available. For example, kitsare available from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit; and Chameleon™ double-stranded, site-directedmutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod described above), Boehringer Mannheim Corp., ClonetechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), NewEngland Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc (e.g., using the Ecksteinmethod above), and Anglian Biotechnology Ltd (e.g., using theCarter/Winter method above).

The above references provide many mutational formats, includingrecombination, recursive recombination, recursive mutation andcombinations or recombination with other forms of mutagenesis, as wellas many modifications of these formats. Regardless of the diversitygeneration format that is used, the nucleic acids of the invention canbe recombined (with each other, or with related (or even unrelated)sequences) to produce a diverse set of recombinant nucleic acids,including, e.g., sets of homologous nucleic acids, as well ascorresponding polypeptides.

A recombinant nucleic acid produced by recombining one or morepolynucleotide sequences of the invention with one or more additionalnucleic acids using any of the above-described formats alone or incombination also forms a part of the invention. The one or moreadditional nucleic acids may include another polynucleotide of theinvention; optionally, alternatively, or in addition, the one or moreadditional nucleic acids can include, e.g., a nucleic acid encoding anaturally-occurring B7-1, co-stimulatory homologue or a subsequencethereof, or any homologous B7-1, co-stimulatory sequence or subsequencethereof (e.g., as found in GenBank or other available literature), or,e.g., any other homologous or non-homologous nucleic acid or fragmentsthereof (certain recombination formats noted above, notably thoseperformed synthetically or in silico, do not require homology forrecombination).

Polypeptides of the Invention

The invention provides isolated or recombinant NCSM polypeptides,fragments thereof, and homologues, variants and derivatives thereof,collectively referred to herein as “NCSM polypeptides” or “NCSMpolypeptide” unless otherwise specifically noted. The term “NCSMpolypeptide” is intended throughout to include amino acid fragments,homologues, derivatives, variants of the polypeptide and proteinsequences specifically disclosed herein unless otherwise noted.Polypeptide variants include those with conservative amino acidsubstations (“conservatively substituted variations”) as describedabove. Also included in this invention are fusion proteins comprisingNCSM polypeptides and proteins, chimeric NCSM polypeptides, comprisingone or more fragments from one or more NCSM polypeptides set forthherein.

As discussed above, the invention provides CD28BP polypeptides andCTLA-4BP polypeptides and fragments of either thereof that bind eitheror both of CD28 or CTLA-4 receptor. In some embodiments, a CD28BPpolypeptide of the invention (including fragments thereof, such assoluble ECDs and ECD fusion proteins, cytoplasmic domains, transmembranedomains, and/or signal peptides, and fusion proteins thereof) has abinding affinity for CD28 that is about equal to or greater than that ofhB7-1 for CD28 (which is about 4×10⁻⁶ M) and/or a binding affinity forCTLA-4 that is about equal to or less than about that of hB7-1 forCTLA-4 (i.e., about 0.2–0.4×10⁻⁶ M). In other embodiments, a CD28BP ofthe invention has a CD28/CTLA-4 binding affinity ratio that is aboutequal to or greater than that of hB7-1. In some such embodiments, aratio of specific binding affinities CD28/CTLA-4 for a CD28BP is atleast about 0.5–1×10⁻¹.

In other embodiments, a CTLA-4BP polypeptide of the invention (includingfragments thereof, such as soluble ECDs and ECD fusion proteins,cytoplasmic domains, transmembrane domains, and/or signal peptides, andfusion proteins thereof) has a CTLA-4/CD28 binding affinity ratio thatis about equal to or greater than that of hB7-1. In some embodiments, aCTLA-4BP polypeptide of the invention has a binding affinity for CTLA-4that is about equal to or greater than that of hB7-1 for CTLA-4 (i.e.,about 4×10⁻⁶ M) and/or a binding affinity for CD28 that is about equalto or less than that of hB7-1 for CD28 (which ranges from about 0.2×10⁻⁶M to about 0.4×10⁻⁶ M). In other embodiments, a CTLA-4BP has a bindingaffinity for CTLA-4 and CD28 that is less than the binding affinity ofhB7-1 for either receptor; however, the ratio of these bindingaffinities ratio (CTLA-4/CD28) is still at least equal to or greaterthan that of hB7-1. In other embodiments, for a CTLA-4BP, a ratio ofspecific binding affinities CTLA-4/CD28 is at least about 10. Alsoincluded are nucleic acid sequences (e.g., DNA and RNA) that encode allaforementioned NCSM polypeptides and fragments thereof having one ormore properties outlined above, vectors comprising such nucleic acidsequences, cells transformed with such vectors.

CD28BP Polypeptides

In one aspect, the invention provides an isolated or recombinant CD28BPpolypeptides comprising an extracellular domain (ECD) sequence, whereinthe ECD sequence has at least about 65%, 70% 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more amino acidsequence identity to an extracellular domain amino acid sequence of atleast one of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, and is nota naturally-occurring extracellular domain amino acid sequence, andwherein said polypeptide has a CD28/CTLA-4 binding affinity ratio aboutequal to or greater than the CD28/CTLA-4 binding affinity ratio of humanB7-1.

For some such polypeptides, the polypeptide comprises an extracellulardomain amino acid sequence or the full-length amino acid sequence of anyone of SEQ ID NOS:48–68, 174–182, 184–221, 283–285, and 290–293. Somesuch polypeptides comprise an extracellular domain amino acid sequenceof any one of SEQ ID NOS:48–68 and 174–209. For some such polypeptides,the polypeptide comprises an extracellular domain amino acid sequenceencoded by a coding polynucleotide sequence that is selected from thegroup of: (a) an ECD coding sequence of a polynucleotide sequenceselected from any of SEQ ID NOS:1–21 and 95–142; (b) an polynucleotidesequence that encodes the ECD of a polypeptide selected from any of SEQID NOS:48–68, 174–221, 283–285, and 290–293; and (c) a polynucleotidesequence which, but for codon degeneracy, hybridizes under stringentconditions over substantially the entire length of a polynucleotidesequence (a) or (b).

Some such CD28BP polypeptides described above have an equal or enhancedbinding affinity for CD28 as compared to a binding affinity of a WTco-stimulatory molecule for CD28. Some such polypeptides have aCD28/CTLA-4 binding affinity ratio at least equal to or greater than theCD28/CTLA-4 binding affinity ratio of hB7-1. In one aspect, some suchpolypeptides have a decreased or a lowered binding affinity for CTLA-4as compared to a binding affinity of a wild type co-stimulatory moleculefor CTLA-4. Some such CD28BP polypeptides may induce T-cellproliferation or T-cell activation or both T-cell proliferation andT-cell activation, such as, e.g., in association with co-stimulation ofT cell receptor/CD3 (by, e.g., an antigen or anti-CD3 antibody). Theinduced T-cell proliferative response may be about equal to or greaterthan that of human B7-1 for some such polypeptides. In another aspect,some such polypeptides described above modulate T-cell activation, butdo not induce proliferation of purified T-cells activated by solubleanti-CD3 mAbs.

In another aspect, the invention provides isolated or recombinant CD28BPpolypeptides that comprise a non-naturally-occurring amino acid sequenceencoded by a nucleic acid comprising a polynucleotide sequence selectedfrom the group of: (a) a polynucleotide sequence selected from SEQ IDNOS:1–21 and 95–142, or a complementary polynucleotide sequence thereof;(b) a polynucleotide sequence encoding a polypeptide selected from SEQID NOS:48–68, 174–221, 283–285, and 290–293, or a complementarypolynucleotide sequence thereof; (c) a polynucleotide sequence which,but for degeneracy of the genetic code, hybridizes under highlystringent conditions over substantially the entire length ofpolynucleotide sequence (a) or (b); (d) a polynucleotide sequencecomprising all or a fragment of (a), (b), or (c), wherein the fragmentencodes a polypeptide having a CD28/CTLA-4 binding affinity ratio aboutequal to or greater than the CD28/CTLA-4 binding affinity ratio of humanB7-1; (e) a polynucleotide sequence encoding a polypeptide, thepolypeptide comprising an amino acid sequence which is substantiallyidentical over at least about 150 contiguous amino acid residues of anyone of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293; and (f) apolynucleotide sequence encoding a polypeptide that has a CD28/CTLA-4binding affinity ratio equal to, about equal to or greater than theCD28/CTLA-4 binding affinity ratio of human B7-1, which polynucleotidesequence has at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identity to at least onepolynucleotide sequence of (a), (b), (c), or (d). In one aspect, suchCD28BP polypeptides comprise the full-length amino acid sequence of anyone of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293.

The above-described polypeptides have a CD28/CTLA-4 binding affinityratio about equal to, equal to or greater than the CD28/CTLA-4 bindingaffinity ratio of human B7-1. Some such polypeptides induce a T-cellproliferation in association with TCR stimulation; the response may beabout equal to or greater than that of human B7-1.

In yet another embodiment, the invention provides isolated orrecombinant polypeptides comprising an amino acid sequence according tothe formula:

MGHTM-X6-W-X8-SLPPK-X14-PCL-X18-X19-X20-QLLVLT-X27-LFYFCSGITPKSVTKRVKETVMLSCDY-X55-TSTE-X60-LTSLRIYW-X69-KDSKMVLAILPGKVQVWPEYKNRTITDMNDN-X101-RIVI-X106-ALR-X110-SD-X113-GTYTCV-X120-QKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVP-X149-X150-X151-DLGNPSPNIRRLICS-X167-X168-X169-GFPRPHL-X177-WLENGEELNATNTT-X192-SQDP-X197-T-X199-LYMISSEL-X208-FNVTNN-X215-SI-X218-CLIKYGEL-X227-VSQIFPWSKPKQEPPIDQLPF-X249-VIIPVSGALVL-X261-A-X263-VLY-X267-X268-ACRH-X273-ARWKRTRRNEETVGTERLSPIYLGSAQSSG (SEQ ID NO:284), or a subsequence thereof comprising theextracellular domain, wherein position X6 is Lys or Glu; position X8 isArg or Gly; position X14 is Arg or Cys; position X18 is Trp or Arg;position X19 is Pro or Leu; position X20 is Ser or Pro; position X27 isAsp or Gly; position X55 is Asn or Ser; position X60 is Glu or Lys;position X69 is Gln or Arg; position X101 is Pro or Leu; position X106is Leu or Gln; position X110 is Pro or Leu; position X113 is Lys or Ser;position X120 is Val or Ile; position X124 is Val or Asp; position X135is Thr or Ala; position X149 is Thr, Ser, or del; position X150 is Ileor del; position X151 is Asn or Thr; position X167 is Thr or del;position X169 is Ser or del; position X169 is Gly or del; position X177is Cys or Tyr; position X192 is Val or Leu; position X197 is Gly or Glu;position X199 is Glu or Lys; position X208 is Gly or Asp; position X215is His or Arg; position X218 is Ala or Val; position X227 is Ser or Leu;position X249 is Trp, Leu, or Arg; position X261 is Ala or Thr; positionX263 is Val, Ala, or Ile; position X267 is Arg or Cys; position X268 isPro or Leu; and position X273 is Gly or Val. Some such polypeptides haveone or more of the properties of CD28 polypeptides described herein,including an ability to enhance an immune response, induce a T cellactivation or proliferation response, exhibit a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than that of hB7-1, and/oralter cytokine production. For some such polypeptides, the induced Tcell response is about equal to or greater than that of hB7-1. In oneembodiment, some such polypeptides comprise an extracellular domainamino acid sequence of any one of SEQ ID NOS:51–56, 58, 61, 66, 67,174–179, 181, 185–187, 189, 192–194, 197, 199, 202, 205, 208, 215, 217,220, and 285.

In another embodiment, some such polypeptides comprise two, three, four,five, six, eight, ten, or more of: Lys at position X6; Arg at positionX8; Arg at position X14; Trp at position X18; Pro at position X19; Serat position X20; Asp at position X27; Asn at position X55; Leu atposition X106; Pro at position X110; Lys at position X113; Val atposition X120; Val at position X124; Thr at position X135; Asn atposition X151; Cys at position X177; Val at position X192; Gly atposition X197; Glu at position X199; Gly at position X208; His atposition X215; Ala at position X218; Trp at position X249; Ala atposition X261; Val at position X263; Arg at position X267; Pro atposition X268; and Gly at position X273. In a preferred embodiment, somesuch polypeptides comprise two, three, four, five, six, eight, ten, ormore of: Arg at position X8; Arg at position X14; Trp at position X18;Pro at position X19; Ser at position X20; Pro at position X110; Val atposition X120; Val at position X124; Cys at position X177; Val atposition X192; Gly at position X197; Glu at position X199; Gly atposition X208; His at position X215; Ala at position X218; Trp atposition X249; Ala at position X261; and Val at position X263. In yetanother preferred embodiment, some such polypeptides comprise theextracellular domain amino acid sequence of SEQ ID NO:66 or SEQ IDNO:285. In yet another preferred embodiment, some such polypeptidescomprise the sequence of SEQ ID NO:66 or SEQ ID NO:285.

In another aspect, the invention provides isolated or recombinant CD28BPpolypeptides comprising a subsequence of an amino acid sequence setforth in any of SEQ ID NOS:48–68, 174–182, 184–221, 283–285, and290–293, wherein the subsequence is the extracellular domain of saidamino acid sequence.

The invention also provides isolated or recombinant polypeptidescomprising an amino acid sequence according to the formula:

MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVM-X50-SCDY-X55-X56-STEELTSLRIYWQKDSKMVL AILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSD-X113-GTYTCV-X120-QK-X123-X124-X125-X126-G-X128-X129-X130-X131-EHL-X135-SV-X138-L-X140-IRADFPVPSITDIGHPAPNVKRIRCSASG-X170-FPEPRLAWMEDGEELNAVNTTV-X193-X194-X195-LDTELYSVSSELD-X209-N-X211-TNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVI-X252-X253-VSGALVLTAVVLYCLACRHVAR (SEQ ID NO:290), orsubsequence thereof comprising the extracellular domain, whereinposition X50 is Leu or Pro; position X55 is Asn or Ser; position X56 isAla or Thr; position X113 is Ser or Lys; position X120 is Ile or Val;position X123 is Pro or deleted; position X124 is Val, Asn, or Asp;position X125 is Leu or Glu; position X126 is Lys or Asn; position X128is Ala or Ser; position X129 is Tyr or Phe; position X130 is Lys or Arg;position X131 is Leu or Arg; position X135 is Ala or Thr; position X138is Arg or Thr; position X140 is Met or Ser; position X170 is Asp or Gly;position X193 is Asp or is deleted; position X194 is Gln or is deleted;position X195 is Asp or is deleted; position X211 is Val or Ala;position X252 is Ile or Val; and position X253 is Leu or Pro. Some suchpolypeptides have at least one of the properties of CD28 polypeptidesdescribed herein, including an ability to enhance an immune response,induce T cell activation or proliferation, exhibit a CD28/CTLA-4 bindingaffinity ratio about equal to or greater than that of hB7-1, and/oralter cytokine production. For some such polypeptides, the induced Tcell response is about equal to or greater than that of hB7-1.

In a preferred embodiment, some such CD28BP polypeptides comprise two,three, four, five, six, eight, ten, or more of: Leu at position X50; Asnat position X55; Ala at position X56; Ser at position X113; Ile atposition X120; Pro at position X123; Val at position X124; Leu atposition X125; Lys at position X126; Ala at position X128; Tyr atposition X129; Lys at position X130; Leu at position X131; Ala atposition X135; Arg at position X138; Met at position X140; Asp atposition X170; Asp at position X193; Asp at position X194; Asp atposition X195; Val at position X211; Ile at position X252; and Leu atposition X253. In yet another preferred embodiment, some suchpolypeptides comprise a sequence of any one of SEQ ID NOS:59, 62, 180,184, 188, 195, 196, 200, 201, 204, 211, 213, 219, and 291.

In another aspect, the invention provides isolated or recombinantpolypeptides comprising an amino acid sequence according to the formula:

MGHTMKWG-X9-LPPKRPCLWLSQLLVLTGLFYFCSG-X35-TPKSVTKRVKETVMLSCDY-X55-TSTEELTSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALR-X110-SDSGTYTCVIQKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENG-X183-ELNATNTT-X192-SQDPETKLYMISSELDFN-X211-TSN-X215-X216-X217-LCLVKYGDLTVSQ-X231-FYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEV-X288-M-X290-SCSQSP (SEQ ID NO:292), or asubsequence thereof comprising the extracellular domain, whereinposition X9 is Thr or Ser; position X35 is Ile or Thr; position X55 isAsn or Ser; position X110 is Leu or Pro; position X124 is Asp or Val;position X135 is Thr or Ala; position X183 is Lys or Glu; position X192is Leu or Val; position X211 is Met or Thr; position X215 is His or isdeleted; position X216 is Ser or is deleted; position X217 is Phe or isdeleted; position X231 is Thr or Ser; position X288 is Lys or Glu;position X290 is Glu or Gln, and wherein said sequence is a nonnaturally-occurring sequence. Further, some such polypeptides have atleast one of the properties of CD28 polypeptides described herein,including an ability to enhance an immune response, induce T cellactivation or proliferation, exhibit a CD28/CTLA-4 binding affinityratio about equal to or greater than that of hB7-1, and/or altercytokine production. For some such polypeptides, the induced T cellresponse is about equal to or greater than that of hB7-1.

In a preferred embodiment, some such polypeptides comprise two, three,four, five, six, eight, ten, or more of the following amino acids: Thrat position X9; Ile at position X35; Asn at position X55; Leu atposition X110; Asp at position X124; Thr at position X135; Lys atposition X183; Leu at position X192; Met at position X211; His atposition X215; Ser at position X216; Phe at position X217; Thr atposition X231; Lys at position X288; and Glu at position X290. In yetanother preferred embodiment, some such polypeptides comprise a sequenceof any one of SEQ ID NOS:48, 182, 183, 212, 214, 216, 218, 221, and 293.

In another aspect, the invention provides isolated or recombinantpolypeptides comprising an amino acid sequence corresponding to anextracellular domain, wherein said amino acid sequence has at leastabout 92% or about 95% amino acid sequence identity to the amino acidsequence corresponding to the extracellular domain of SEQ ID NO:66, andwherein said polypeptide has a CD28/CTLA-4 binding affinity ratiogreater than the CD28/CTLA-4 binding affinity ratio of human B7-1 or anability to induce a T cell proliferation or activation response that isgreater than or about equal to that induced by hB7-1. Some such isolatedor recombinant polypeptides further comprise at least one further aminoacid sequence corresponding to a signal peptide. Some such isolated orrecombinant polypeptides further comprise at least one further aminoacid sequence corresponding to a transmembrane domain or a cytoplasmicdomain.

The invention also provides isolated or recombinant polypeptidevariants, each polypeptide comprising an amino acid sequence thatdiffers from the ECD amino acid sequence of B7-1 polypeptide of anArtiodactyla mammal, such as a bovine B7-1, wherein the differencebetween the amino acid sequence of the variant and the ECD amino acidsequence of the Artiodactyla mammal B7-1 (e.g., bovine B7-1) comprises adifferent amino acid at one or more of the following amino acid residuepositions: position 110, 124, 135, 192, 197, 199, 211, 213, 217, 218,221, 225, 227, 231, 233, 235, 236, 237, 239, 240, 242, 243, and 244,wherein each position corresponds to the position in the amino acidsequence of the bovine B7-1 of SEQ ID NO:280. An Artiodactyla mammalincludes cloven-hoofed mammals, such as bovine, sheep, goats, camels,pigs, deer, giraffes, and antelope(www.ucmp.berkeley.edu/mammal/eutheria/ungulate.html). Some suchpolypeptide variants comprise variants of full-length bovine B7-1polypeptide.

For some such polypeptide variants, the difference between the aminoacid sequence of the variant and the ECD amino acid sequence of theArtiodactyla B7-1 (e.g., bovine B7-1) comprises at least one of: (a) adifferent amino acid at position 110 which is Pro; (b) a different aminoacid at position 124 which is Val; (c) a different amino acid atposition 135 which is Ala; (d) a different amino acid at position 192which is Val; (e) a different amino acid at position 197 which is Gly;(f) a different amino acid at position 199 which is Glu; (g) a differentamino acid at position 211 which is Val; (h) a different amino acid atposition 213 which is Asn; (i) a different amino acid at position 217which is Ile; (j) a different amino acid at position 218 which is Val;(k) a different amino acid at position 221 which is Ile; (1) a differentamino acid at position 225 which is Glu; (m) a different amino acid atposition 227 which is Ser; (n) a different amino acid at position 231which is Ile; (o) a different amino acid at position 233 which is Pro;(p) a different amino acid at position 235 which is Ser; (q) a differentamino acid at position 236 which is Lys; (r) a different amino acid atposition 237 which is Pro; (s) a different amino acid at position 239which is Gln; (t) a different amino acid at position 240 which is Glu;(u) a different amino acid at position 242 which is Pro; (v) a differentamino acid at position 243 which is Ile; and (w) a different amino acidat position 244 which is Asp.

For some such polypeptide variants, the difference between the aminoacid sequence of the variant and the ECD amino acid sequence of theArtiodactyla B7-1 (e.g., bovine B7-1) further comprises at least one of:(1) a different amino acid at position 246 which is Leu; (2) a differentamino acid at position 247 which is Pro; (3) a different amino acid atposition 248 which is Phe; (4) a different amino acid at position 250which is Val; and (5) a different amino acid at position 253 which isPro, wherein each position corresponds to the position in the amino acidsequence of bovine B7-1 of SEQ ID NO:280. Some such polypeptide variantsfurther comprise a signal peptide (e.g., of a WT mammalian B7-1 or NCSMpolypeptide described herein). Some such polypeptide variants alsocomprise a transmembrane domain and/or cytoplasmic domain, including,e.g., a TMD and/or CD of a WT mammalian B7-1 or NCSM polypeptidedescribed herein.

In some such variants of bovine B7-1, the amino acid in SEQ ID NO:278 atat least one of positions 254, 255, and 256 is deleted. Some such bovineB7-1 variants further comprise at least one of: (6) a different aminoacid at position 257 which is Val; (7) a different amino acid atposition 258 which is Ser; (8) a different amino acid at position 260which is Ala; (9) a different amino acid at position 261 which is Leu;(10) a different amino acid at position 263 which is Leu; (11) adifferent amino acid at position 264 which is Thr; (12) a differentamino acid at position 267 which is Val; (13) a different amino acid atposition 269 which is Tyr; (14) a different amino acid at position 272which is Ala; (15) a different amino acid at position 275 which is His;(16) a different amino acid at position 276 which is Val; (17) adifferent amino acid at position 275 which is His; wherein each positioncorresponds to the position in the amino acid sequence of SEQ ID NO:280.

Some such bovine B7-1 variants further comprise at least one of: (18) adifferent amino acid at position 276 which is Val; (19) a differentamino acid at position 278 which is Arg; (20) a different amino acid atposition 279 which is Trp; (21) a different amino acid at position 280which is Lys; (22) a different amino acid at position 281 which is Arg;(23) a different amino acid at position 282 which is Thr; (24) adifferent amino acid at position 284 which is Arg; (25) a differentamino acid at position 287 which is Glu; (26) a different amino acid atposition 288 which is Thr; (27) a different amino acid at position 289which is Val; (28) a different amino acid at position 290 which is Gly;(29) a different amino acid at position 291 which is Thr; (30) adifferent amino acid at position 292 which is Glu; (31) a differentamino acid at position 293 which is Arg; (32) a different amino acid atposition 294 which is Leu; wherein each position corresponds to theposition in the amino acid sequence of SEQ ID NO:280.

Some of the above-described polypeptide variants of Artiodactylamammalian B7-1 (e.g., bovine B7-1) further comprise the following aminoacid sequence at the C terminus: IYLGSAQSSG (SEQ ID NO:320).

Some of the above-described polypeptide variants of Artiodactylamammalian B7-1 (e.g., bovine B7-1) exhibit a CD28/CTLA-4 bindingaffinity ratio that is equal to or greater than that of hB7-1 and/orhave an ability to induce a T cell proliferation or activation responseabout equal to or greater than that induced by hB7-1.

The invention also includes nucleic acid sequences (e.g., DNA and RNA)that encode all aforementioned CD28BP polypeptides and Artiodactylapolypeptide variants, and fragments thereof having one or more of theproperties described above, and complementary nucleic acid sequencesthereof; and expression vectors comprising all such nucleic acidsequences, and cells transformed with such expression vectors.

CTLA-4BP Polypeptides

In one aspect, the invention provides isolated or recombinant CTLA-4BPpolypeptides each comprising an amino acid sequence having at leastabout 85%, 88%, 89%, 90%, 91%, 92&, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or more percent identity to at least one of SEQ ID NOS:69–92,222–252, 286–289, or a subsequence thereof comprising the extracellulardomain, wherein said sequence (a) is a non naturally-occurring sequence,and (b) comprises at least one of: Gly at position 2; Thr at position 4;Arg at position 5; Gly at position 8; Pro at position 12; Met atposition 25; Cys at position 27; Pro at position 29; Leu at position 31;Arg at position 40; Leu at position 52; His at position 65; Ser atposition 78; Asp at position 80; Tyr at position 87; Lys at position120; Asp at position 122; Lys at position 129; Met at position 135; Pheat position 150; Ile at position 160; Ala at position 164; His atposition 172; Phe at position 174; Leu at position 176; Asn at position178; Asn at position 186; Glu at position 194; Gly at position 196; Thrat position 199; Ala at position 210; His at position 212; Arg atposition 219; Pro at position 234; Asn at position 241; Leu at position244; Thr at position 250; Ala at position 254; Tyr at position 265; Argat position 266; Glu at position 273; Lys at position 275; Ser atposition 276; an amino acid deletion at position 276; or Thr at position279, wherein the position number corresponds to that of hB7-1 amino acidsequence (SEQ ID NO:278), wherein said polypeptide has a CTLA-4/CD28binding affinity ratio about equal to or greater than a CTLA-4/CD28binding affinity ratio of hB7-1.

Such CTLA-4BP polypeptides described above have an altered bindingaffinity for CTLA-4 and/or CD28 as compared to the binding affinity of aWT co-stimulatory molecule for CD28 or CTLA-4. Some such polypeptideshave a CTLA-4/CD28 binding affinity ratio about equal to or greater thanthat of hB7-1. In one aspect, some such polypeptides have a decreasedbinding affinity for CD28 or CTLA-4 as compared to a binding affinity ofa hB7-1 to CD28 or CTLA-4, respectively. Some such polypeptides mayinhibit at least one or both of T-cell proliferation or activation inassociation with co-stimulation of TCR/CD3 (by, e.g., an antigen oranti-CD3 antibody). The induced T-cell proliferative response may beless than that of hB7-1 for some such polypeptides. In another aspect,some such polypeptides described above modulate T-cell activation, butdo not induce proliferation of purified T-cells activated by solubleanti-CD3 mAbs.

Some such CTLA-4BP polypeptides each comprise an amino acid sequencehaving at least about 98% identity to at least one of SEQ ID NOS:69–92,222–252, and 286–289, said sequence comprising at least one of: Gly atposition 2; Gly at position 8; Cys at position 27; His at position 65;Asp at position 80; Asp at position 122; Met at position 135; Phe atposition 150; Ala at position 164; Phe at position 174; Asn at position186; Glu at position 194; Arg at position 219; Thr at position 250; Argat position 266; Lys at position 275; and Ser at position 276, whereinamino acid position numbers correspond to that of the hB7-1 amino acidsequence (SEQ ID NO:278). In one aspect, such polypeptides may comprisethe ECD or full-length amino acid sequence of any one of SEQ IDNOS:69–92, 222–252, and 286–289.

In a preferred embodiment, some such above-described CTLA-4BPpolypeptides comprise an amino acid sequence having at least about 98%identity to the extracellular domain of at least one of SEQ IDNOS:69–92, 222–252, and 286–289, said sequence comprising at least oneof: His at position 65; Asp at position 80; Asp at position 122; Met atposition 135; Phe at position 150; Ala at position 164; Phe at position174; Asn at position 186; Glu at position 194; and Arg at position 219,wherein the amino acid position numbers correspond to that of hB7-1amino acid sequence (SEQ ID NO:278).

Further, some such polypeptides comprise a sequence having at leastabout 98% identity to the ECD of at least one of SEQ ID NOS:69–92,222–252, 286–289, said sequence comprising at least two, three, four,five, six or more of: His at position 65; Asp at position 80; Asp atposition 122; Met at position 135; Phe at position 150; Ala at position164; Phe at position 174; Asn at position 186; Glu at position 194; andArg at position 219, wherein the amino acid position numbers correspondto that of h B7-1 amino acid sequence (SEQ ID NO:278). Some suchCTLA-4BP polypeptides comprise an ECD amino acid sequence of any one ofSEQ ID NOS:69–92 and 222–247. In a preferred embodiment, such CTLA-4BPpolypeptides comprise an ECD sequence of any one of SEQ ID NOS:81, 85,86, 88, 90, and 91.

In another aspect, some such above-described CTLA-4BP polypeptidescomprises an ECD domain sequence encoded by a coding polynucleotidesequence, the coding polynucleotide sequence selected from the group:(a) an ECD coding sequence of a polynucleotide sequence selected fromany of SEQ ID NOS:22–45 and 143–173; (b) a polynucleotide sequence thatencodes the ECD amino acid sequence of a polypeptide selected from anyof SEQ ID NOS:69–92, 222–252, and 286–289; and (c) a polynucleotidesequence which, but for the codon degeneracy, hybridizes under stringentconditions over substantially the entire length of a polynucleotidesequence (a) or (b).

Some such above-described isolated or recombinant polypeptides,including, but not limited to, those having the binding and/orproliferation properties discussed above, further comprise a signalpeptide amino acid sequence encoded by a signal peptide codingnucleotide sequence, the signal peptide coding nucleotide sequenceselected from the group of: (a) a nucleotide sequence comprising anucleotide fragment of a polynucleotide sequence selected from any ofthe group of SEQ ID NOS:22–45 and 143–173, wherein said nucleotidefragment encodes a signal peptide; (b) a nucleotide sequence thatencodes the signal peptide of a polypeptide selected from any of thegroup of SEQ ID NOS:69–92, 222–252, and 286–289; and (c) a nucleotidesequence which, but for codon degeneracy, hybridizes under at leaststringent conditions over substantially the entire length of anucleotide sequence (a) or (b).

Some of the above-described isolated or recombinant polypeptides furthercomprising a transmembrane domain (TMD) amino acid sequence encoded by aTMD nucleotide sequence selected from the group of: (a) a nucleotidesequence of a polynucleotide sequence selected from any of the group ofSEQ ID NOS:22–45 and 143–173, wherein said nucleotide sequence encodes aTMD polypeptide; (b) a nucleotide sequence that encodes the TMD of apolypeptide selected from any of the group of SEQ ID NOS:69–92, 222–252,and 286–289; and (c) a nucleotide sequence which, but for codondegeneracy, hybridizes under at least stringent conditions oversubstantially the entire length of a nucleotide sequence (a) or (b).Some such polypeptides further comprise a cytoplasmic domain (CD) aminoacid sequence encoded by a CD nucleotide sequence selected from thegroup of: (a) a nucleotide sequence of a polynucleotide sequenceselected from any of the group of SEQ ID NOS:22–45 and 143–173, whereinsaid nucleotide sequence encodes a CD polypeptide; (b) a nucleotidesequence that encodes the CD of a polypeptide selected from any of thegroup of SEQ ID NOS:69–92, 222–252, and 286–289, and 290–293; and (c) anucleotide sequence which hybridizes under at least stringent conditionsover substantially the entire length of a nucleotide sequence (a) or(b).

The invention includes isolated or recombinant polypeptides eachcomprising an amino acid sequence that differs from a primate B7-1sequence in at least one of the mutation selected from the following atan amino acid residue position of indicated, wherein the positioncorresponds to the amino acid position within the amino acid sequence ofhB7-1 as shown in SEQ ID NO:278: 1) 40 Arg; 52 Leu; 65 His; 122 Asp; 129Lys; 135 Met; 164 Ala; 174 Phe; 196 Gly; 199 Thr; 210 Ala; 219 Arg; 234Pro; 241 Asn; or 2) 12 Pro; 25 Met; 27 Cys; 29 Pro; 40 Arg; 52 Leu; 65His; 122 Asp; 129 Lys; 135 Met; 164 Ala; 174 Phe; 196 Gly; 199 Thr; 210Ala; 219 Arg; 234 Pro; 241 Asn; 254 Ala; 275 Lys; 276 Ser; or 279 Thr.The invention also provides isolated or recombinant polypeptides eachcomprising an amino acid sequence that differs from a primate B7-1sequence in at least one mutation selected from: Ser 12 Pro; Leu 25 Met;Gly 27 Cys; Ser 29 Pro; Lys 40 Arg; His 52 Leu; Tyr 65 His; Glu 122 Asp;Glu 129 Lys; Thr 135 Met; Thr 164 Ala; Ser 174 Phe; Glu 196 Gly; Ala 199Thr; Thr 210 Ala; Lys 219 Arg; Thr 234 Pro; Asp 241 Asn; Val 254 Ala;Arg 275 Lys; Arg 276 Ser; or Arg 279 Thr. The mutation being indicatedis relative, e.g., to human B7-1 with the amino acid sequence shown inSEQ ID NO:278, the sequence does not occur in nature, and, in somesequences, the polypeptide has a CTLA-4/CD28 binding affinity ratioabout equal to, equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of human B7-1. Each amino acid position corresponds tothe position of the amino acid sequence of SEQ ID NO:278. In someembodiments, the sequence of some such polypeptides differs from primateB7-1 sequence in at least two of said mutations. In some aspects, theprimate B7-1 is hB7-1 (SEQ ID NO:278), and in some aspects, the sequencediffers from the hB7-1 sequence in at least two mutations.

In another aspect, the invention provides isolated or recombinantCTLA-4BP polypeptides comprising an amino acid sequence having at leastabout 75%, 80%, 85%, 90%, 95%, or more percent identity to at least oneof SEQ ID NOS:263–272, or a subsequence thereof comprising the ECD,wherein the sequence is not a naturally-occurring, and the polypeptidehas a CTLA-4/CD28 binding affinity ratio about equal to or greater thanthe CTLA-4/CD28 binding affinity ratio of hB7-1 or an ability to inducea T cell proliferation or activation response about equal to or lessthan that of hB7-1.

In yet another aspect, the invention provides isolated or recombinantpolypeptides that each comprise a non naturally-occurring amino acidsequence encoded by a nucleic acid comprising a polynucleotide sequenceselected from: (a) a polynucleotide sequence selected from SEQ IDNOS:22–45, 143–173, 253–262, or a complementary polynucleotide sequencethereof; (b) a polynucleotide sequence encoding a polypeptide selectedfrom SEQ ID NOS:69–92, 222–247, 263–272, 286–289, or a complementarypolynucleotide sequence thereof; (c) a polynucleotide sequence whichhybridizes under at least stringent or highly stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b);(d) a polynucleotide sequence comprising all or a fragment of (a), (b),or (c), wherein the fragment encodes a polypeptide having a CTLA-4/CD28binding affinity ratio about equal to or greater than that of hB7-1; (e)a polynucleotide sequence encoding a polypeptide, the polypeptidecomprising an amino acid sequence which is substantially identical overat least about 150, 180, 200, 225, 250 or more contiguous amino acidresidues of any one of SEQ ID NOS:69–92, 222–247, 263–272, 286–289, and(f) a polynucleotide sequence encoding a polypeptide that has aCTLA-4/CD28 binding affinity ratio about equal to or greater than thatof hB7-1, which polynucleotide sequence has at least about 70%, 80%,85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or more identity to at least onepolynucleotide sequence of (a), (b), (c), or (d). Some such polypeptidescomprise an amino acid sequence of any one of SEQ ID NOS:69–92, 222–247,263–272, and 286–289.

Such above-described polypeptides have a CTLA-4/CD28 binding affinityratio about equal to or greater than the CTLA-4/CD28 binding affinityratio of human B7-1. Some such polypeptides inhibit T-cellproliferation. The induced T-cell response may be less than that ofhuman B7-1.

In yet another aspect, the invention includes isolated or recombinantpolypeptides that each comprise a sequence according to the formula:

MGHTRRQGTSP-X12-KCPYLKFFQLLV-X25-ACL-X29-HLCSGVIHVT-X40-EVKEVATLSCGLNVSVEELAQTRIHWQKEKKMVLTMMSGDMNIMWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKY-X122-KDAFKR-X129-HLAEVMLSVKADFPTPSITDFEIPPSNIRRIICS-X164-SGGFPEPHLFWLENGEELNAINTTVSQDPET-X196-LYTVSSKLDFNMTANHSFMCLI-X219-YGHLRVNQTFNWNTPKQEHFP-X241-NLLPSWAITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV (SEQ ID NO:287), or asubsequence thereof comprising the extracellular domain, whereinposition X12 is Ser or Pro; position X25 is Leu or Met; position X29 isSer or Pro; position X40 is Lys or Arg; position X122 is Glu or Asp;position X129 is Glu or Lys; position X164 is Thr or Ala; position X196is Glu or Gly; position X219 is Lys or Arg; and position X241 is Asp orAsn. In a preferred embodiment, some such polypeptides comprise the ECDof SEQ ID NO:288 or SEQ ID NO:289. In another preferred embodiment, somesuch polypeptides comprise the amino acid sequence SEQ ID NO:288 or SEQID NO:289. In one aspect, such polypeptides exhibit at least one of theCTLA-4BP properties described above, including a CTLA-4/CD28 bindingaffinity ratio about equal to or greater than the CTLA-4/CD28 bindingaffinity ratio of human B7-1. Some such polypeptides inhibit T-cellproliferation; for some polypeptides, the induced T-cell response isless than that induced by hB7-1 in the presence of e.g., anti-CD3 Abs orantigen.

The invention also provides isolated or recombinant polypeptides thateach comprise a subsequence of an amino acid sequence set forth in anyof SEQ ID NOS:69–92, 222–247, 263–272, and 286–289, wherein thesubsequence is the ECD of the amino acid sequence.

In addition, the invention provides novel isolated or recombinantpolypeptides corresponding to baboon and orangutan B7-1. Suchpolypeptides comprise the sequence SEQ ID NO:93 or SEQ ID NO:94, or asubsequence thereof, wherein the subsequence comprises at least one of:the signal sequence, extracellular domain, transmembrane domain, and thecytoplasmic domain of the polypeptide.

Also included are nucleic acid sequences (e.g., DNA and RNA) that encodeall aforementioned CTLA-4BP polypeptides and fragments thereof havingone or more of the properties described above, and expression vectorscomprising such nucleic acid sequences, and cells transformed with suchexpression vectors.

B7-1 and B7-2 Polypeptide Variants

The invention also provides polypeptide variants of a WT or mutant B7-1polypeptide, including, e.g., polypeptide variants of the hB7-1polypeptide shown in SEQ ID NO:278 or other parental primate B7-1polypeptide described herein (e.g., SEQ ID NOS:93–94, 279–282). Eachsuch variant comprises an amino acid sequence that differs from theamino acid sequence of the WT or mutant (reference) B7-1 by at least oneamino acid residue. In one aspect, the polypeptide variant comprises anamino acid sequence that differs from that of a full-length WT or mutant(reference) B7-1 polypeptide by substitution in said full-length B7-1polypeptide of at least one amino acid residue at a position thatcorresponds to position 65 of the hB7-1 polypeptide sequence shown inSEQ ID NO:278. The substituted amino acid residue at this position maycomprise any amino acid residue that differs from that in the referencesequence. In another aspect, the substituted amino acid at this positionis any amino acid residue other than alanine or an amino acid residueexisting at that position in a known primate B7-1 polypeptide sequence.In another aspect, the substituted amino acid at this position is anamino acid residue selected from the group consisting of histidine(His), arginine (Arg), lysine (Lys), proline (Pro), phenylalanine (Phe),and tryptophan (Trp). Polypeptide variants of the invention includeamino acid sequences that differ from the amino acid sequence of a WT ormutant primate B7-1 polypeptide by at least one amino acid substitution,wherein the at least one amino acid substitution comprises asubstitution in the amino acid sequence of said primate B7-1 for theamino acid position that corresponds to position 65 of SEQ ID NO:278with any amino acid residue selected from the group of His, Arg, Lys,Pro, Phe, and/or Trp. Usually, the substituted amino acid is His, andthe amino acid for which His is substituted is Tyr. Polypeptide variantsalso include amino acid sequences that differ from the amino acidsequence of SEQ ID NO:278 by the substitution of the amino acid residuein SEQ ID NO:278 (e.g., Tyr) at position 65 with any amino acid residueselected from the group of His, Arg, Lys, Pro, Phe, and Trp. Preferably,the polypeptide variant comprises an amino acid sequence that differsfrom that of SEQ ID NO:278 by at least one amino acid substitutioncomprising the substitution of His for Tyr at position 65 in SEQ IDNO:278 (Tyr65His substitution).

The invention also provides polypeptide variants of a polypeptidefragment or segment of a full-length WT or mutant (reference) B7-1polypeptide, including, but not limited to, a primate B7-1 polypeptide.In this aspect, the polypeptide variant comprises an amino acid sequencethat differs from a first amino acid sequence, which comprises an aminoacid fragment or segment of a full-length amino acid sequence of aprimate B7-1, by at least one amino acid residue. The amino acidfragment typically comprises a signal peptide and/or ECD polypeptide ora mature domain of the primate B7-1 amino acid sequence. Some suchpolypeptide variants comprise an amino acid sequence that differs fromsaid first amino acid sequence comprising a signal peptide and/or ECD ora mature domain of a primate B7-1 sequence by substitution in said firstamino acid sequence of at least one amino acid residue at a positionthat corresponds to position 65 of hB7-1 (SEQ ID NO:278). Thesubstituted amino acid at this position may comprise any amino acidresidue that differs from that in the first amino acid sequence.Usually, the substituted amino acid comprises an amino acid other thanalanine or an amino acid residue existing at that position in a primateB7-1 polypeptide sequence. Preferably, the substituted amino acid atthis position comprises an amino acid residue selected from the groupconsisting of His, Arg, Lys, Pro, Phe, and/or Trp.

Also included are polypeptide variants comprising amino acid sequencesthat differ from a first amino acid sequence, wherein the first aminoacid sequence comprises a signal peptide sequence and/or ECD polypeptidesequence of a primate B7-1 polypeptide sequence (including, e.g.,hB7-1), and wherein the variant that differs from said first amino acidsequence by the substitution of at least one amino acid residue in thefirst amino acid sequence at a position corresponding to position 65 ofthe amino acid sequence of SEQ ID NO:278. In one such aspect, thesubstituted amino acid residue is selected from the group of His, Arg,Lys, Pro, Phe, and/or Trp. Usually, the polypeptide variant comprises anamino acid sequence that differs from an amino acid sequence comprisingamino acids 1–243 of SEQ ID NO:278 by the substitution of His for Tyr atposition 65 of SEQ ID NO:278 (Tyr65His substitution).

B7-1 polypeptide variants of a full-length primate B7-1 polypeptide orpolypeptide fragments thereof (e.g., corresponding to a signal peptideand/or ECD or mature domain of a primate B7-1 as described above) havean altered binding activity compared with the binding activity of thefull-length primate B7-1 polypeptide sequence (or polypeptide fragmentthereof as described above) or an altered binding affinity ratiocompared with the binding affinity ratio of the full-length primate B7-1polypeptide sequence (or polypeptide fragment thereof as describedabove). Some such polypeptide variants of a primate B7-1 polypeptide (orfragment thereof described above) do not bind a CD28-Ig (FIGS. 23A–23B)as does hB7-1, but they do bind CTLA-4-Ig in a manner that issubstantially identical or equivalent to that of hB7-1, or in a mannerthat produces a binding profile substantially identical or equivalent tothat of hB7-1. Some such polypeptide variants have a CTLA-4/CD28 bindingaffinity ratio that is about equal to or greater than the CTLA-4/CD28binding affinity ratio of a primate B7-1 (e.g., hB7-1), under theconditions described in FIGS. 23–24. In addition, some such variantsinduce a decreased level of T cell proliferation compared to the levelof T cell proliferation induced by hB7-1 or CD28BP-15 (FIGS. 23A–23B).

The invention includes a polypeptide variant of a WT or mutant B7-1having an altered binding activity or altered binding affinity ratiocompared with the binding activity or binding affinity ratio of a firstB7-1 polypeptide or polypeptide fragment thereof (e.g., a polypeptidefragment corresponding to a signal peptide and/or ECD or mature domainof the first B7-1 polypeptide as described above), wherein thepolypeptide variant has an amino acid sequence that differs from theamino acid sequence of the first B7-1 polypeptide (or polypeptidefragment thereof) and is at least about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical with theamino acid sequence of the full-length hB7-1 polypeptide sequence (i.e.,the second polypeptide) shown in SEQ ID NO:278 or with the amino acidsequence of a polypeptide fragment of SEQ ID NO:278, such as, e.g., afragment corresponding to a signal peptide and/or ECD or mature domain(e.g., a fragment comprising amino acid residues 1–243, 35–243, or35–288 of SEQ ID NO:278, respectively). The difference between the aminoacid sequence of the variant and the amino acid sequence of the firstB7-1 polypeptide (or fragment thereof) comprises a different amino acidat a position corresponding to position 65 of the amino acid sequence ofSEQ ID NO:278. In one aspect, the different amino acid is selected fromthe group of His, Arg, Lys, Pro, Phe, and/or Trp, and, preferably,comprises His. The first B7-1 polypepticle may comprise a WT B7-1 or amutant, derivative, or conservatively substituted variant of the WT B7-1and some such variants induce a decreased level of T cell proliferationor lack T cell proliferation compared to the level of T cellproliferation induced by hB7-1.

The invention provides a polypeptide variant of a WT or mutant B7-2polypeptide, including, but not limited to, e.g., a polypeptide variantof human B7-2 (hB7-2) or other primate B7-2 polypeptide, wherein thevariant comprises an amino acid sequence that differs from the aminoacid sequence of a WT or mutant hB7-2 by at least one amino acidresidue. The full-length polypeptide and nucleic acid sequences of a WT(reference) hB7-2 are provided in GenBank at GenBank Accession Nos.AAA58389 and L25259, respectively (see Freeman, G. J. et al., (1993)Science 262:909–911). The full-length polypeptide and nucleic acidsequences of a second WT (reference) hB7-2 are provided at GenBankAccession Nos. AAA86473 and U17717, respectively (see Azuma, M. et al.,(1993) Nature 366:76–79). In one aspect, the polypeptide variantcomprises an amino acid sequence that differs from that of a full-lengthWT or mutant B7-2 (or fragment thereof comprising a signal peptideand/or ECD or mature domain of the full-length B7-2) polypeptide bysubstitution in said full-length B7-2 polypeptide (or fragment thereof)of at least one amino acid residue at an amino acid positioncorresponding to: 1) position 65 of the hB7-1 polypeptide sequence shownin SEQ ID NO:278; 2) position 56 of the B7-2 polypeptide sequence shownat GenBank Acc. No. AAA58389; or 3) position 50 of the B7-2 polypeptidesequence shown at GenBank Acc. No. AAA86473. Usually, the substitutedamino acid at this position is an amino acid residue selected from thegroup consisting of His, Arg, Lys, Pro, and/or Trp. In some aspect, thesubstituted amino acid replaces phenylalanine in B7-2. The B7-2polypeptide variant may comprise a modified amino acid sequencecomprising the B7-2 amino acid sequence shown at GenBank Acc. No.AAA58389 (or an amino acid fragment thereof comprising a signal peptideand/or ECD or mature domain of said B7-2 sequence) that has beenmodified by a Phe56His substitution. The B7-2 polypeptide variant maycomprise a modified amino acid sequence comprising the B7-2 amino acidsequence shown at GenBank Acc. No. AAA86473 (or an amino acid fragmentthereof comprising a signal peptide and/or ECD or mature domain of saidB7-2 sequence) that has been modified by a Phe50His substitution.

Some such B7-2 polypeptide variants of a full-length primate B7-2polypeptide (or polypeptide fragment thereof, such as, e.g., that whichcorresponds to a signal peptide and/or ECD or mature domain of a primateB7-2) have an altered binding activity compared with the bindingactivity of the full-length (reference) primate B7-2 polypeptidesequence (or polypeptide fragment thereof) or an altered bindingaffinity ratio compared with the binding affinity ratio of thefull-length primate B7-2 polypeptide sequence (or polypeptide fragmentthereof). Some such polypeptide variants of a primate B7-2 polypeptide(or fragment thereof) bind a CTLA-4 receptor with an equal or greaterbinding affinity than does the primate B7-2 polypeptide (or fragmentthereof) and/or bind a CD28 receptor with an equal or lesser bindingaffinity than does the primate B7-2 (or fragment thereof). Some B7-2polypeptide variants have a CTLA-4/CD28 binding affinity ratio that isabout equal to or greater than the CTLA-4/CD28 binding affinity ratio ofa primate B7-2 polypeptide sequence or fragment thereof. Some suchvariants induce a decreased level of T cell proliferation compared tothe level of T cell proliferation induced by hB7-2 and/or CD28BP-15.

Also included is a polypeptide variant of a WT or mutant B7-2 having analtered binding activity or altered binding affinity ratio compared withthe binding activity or binding affinity ratio of a first B7-2polypeptide or polypeptide fragment thereof (e.g., a polypeptidefragment corresponding to a signal peptide and/or ECD or mature domainof the first B7-2 polypeptide), under the conditions described inExample IX (see, e.g., FIGS. 23–24), wherein the B7-2 polypeptidevariant has an amino acid sequence that differs from the amino acidsequence of the first B7-2 polypeptide (or polypeptide fragment thereof)and is at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% homologous or identical with the amino acid sequence ofthe hB7-2 polypeptide sequence (i.e., the second polypeptide) shown atGenBank Acc. No. AAA58389 or AAA86473, or with the amino acid sequenceof a polypeptide fragment of either of such B7-2 sequence shown inGenBank. The difference between the amino acid sequence of the B7-2polypeptide variant and the amino acid sequence of the first B7-2polypeptide (or fragment thereof) comprises a different amino acid at aposition corresponding to: 1) position 65 of the amino acid sequence ofhB7-1 shown in SEQ ID NO:278; 2) position 56 of the hB7-2 polypeptidesequence at GenBank Acc. No. AAA58389; or 3) position 50 of the hB7-2polypeptide sequence at GenBank Acc. No. AAA86473. The different aminoacid may be selected from the group of His, Arg, Lys, Pro, and/or Trp,and preferably comprises His. The first B7-2 polypeptide may comprise aWT B7-2 or a mutant, derivative, or conservatively substituted variantof the WT B7-2. Some variants induce a decreased level of T cellproliferation compared to that induced by hB7-2 and/or CD28BP-15.

Also included are nucleic acid sequences (e.g., DNA and RNA) that encodeall aforementioned B7-1 and B7-2 polypeptide variants and fragmentsthereof having one or more of the properties described above, andexpression vectors comprising such nucleic acid sequences, and cellstransformed with such expression vectors. Degenerate nucleotidesequences of such nucleotide variants are also a part of the invention.Also included are nucleotide variants (e.g., DNA or RNA variants) of anucleic acid sequence encoding a WT or mutant B7-1 polypeptide,including, but not limited to, e.g., a nucleic acid variant of anucleotide sequence encoding a (reference) hB7-1 polypeptide (SEQ IDNO:273) or other parental primate B7-1 described herein (SEQ IDNOS:46–47, 274–277) or a fragment thereof comprising, e.g., a signalpeptide and/or ECD polypeptide or a mature domain of the (reference)human or primate B7-1. Each such variant comprises a nucleic acidsequence that differs from the nucleic acid sequence of a WT or mutant(reference) B7-1 nucleic acid sequence by at least one nucleic acidresidue. The nucleic acid variant may comprise a nucleic acid sequencethat differs from a first nucleic acid sequence encoding a full-lengthWT or mutant B7-1 polypeptide (or polypeptide fragment thereof) bysubstitution in said first nucleic acid sequence of at least onedifferent nucleic acid residue at a position that corresponds to one ofthe three nucleic acid residues in the codon that encodes the amino acidcorresponding to the amino acid at position 65 of the hB7-1 polypeptideof SEQ ID NO:278. The modified codon usually encodes an amino acidselected from the group of His, Arg, Lys, Pro, Phe, and/or Trp, and,typically, His. All codons encoding such amino acids are well known. Inanother aspect, the nucleic acid variant comprises a nucleic acidsequence that differs from a first nucleic acid sequence that encodes afull-length WT or mutant B7-1 polypeptide (or polypeptide fragmentthereof) by at least one nucleic acid substitution in said first nucleicacid sequence, wherein said at least one nucleic acid substitutioncomprises a substitution of a cytosine (C) for a thymine (T) at position193 in SEQ ID NO:273.

In one aspect, the nucleic acid comprises: (a) a polynucleotide sequencecomprising the polynucleotide sequence of SEQ ID NO:273 in which the 3nucleic acid residues TAC at positions 193–195 are replaced by the threenucleic acid residues CAC, or a complementary polynucleotide sequencethereof; (b) a polynucleotide sequence encoding a polypeptide of SEQ IDNO:278, in which the codon comprising 3 nucleotide residues encoding Tyrare replaced by a codon comprising three nucleotide residues encodingHis, or a complementary polynucleotide sequence thereof; and (c) apolynucleotide sequence comprising all or a nucleotide fragment of (a)or (b), wherein the nucleotide fragment encodes a polypeptide having aCTLA-4/CD28 binding affinity ratio about equal to or greater than theCTLA-4/CD28 binding affinity ratio of human B7-1 or a polypeptide havingan ability to induce a T cell proliferation response that is less thanthat induced by human B7-1.

In addition, the invention includes nucleic acid variants (e.g., DNA orRNA variants) of a nucleic acid sequence encoding a WT or mutant B7-2polypeptide, including, but not limited to, a nucleic acid variant ofthe (reference) nucleotide sequence shown at GenBank Acc. No. AAA86473and U17717, or a nucleic acid variant of a nucleotide sequence encodinga (reference) hB7-2 polypeptide (GenBank Acc. No. AAA58389 or AAA86473)or other primate B7-2 sequence, or a polypeptide fragment thereofcomprising, e.g., a signal peptide and/or ECD polypeptide or a maturedomain of the (reference) human or primate B7-2. Each such variantcomprises a nucleic acid sequence that differs from the nucleic acidsequence of a WT or mutant (reference) B7-2 nucleic acid sequence by atleast one nucleic acid residue. The nucleic acid variant may comprise anucleic acid sequence that differs from a first nucleic acid sequenceencoding a full-length WT or mutant B7-2 polypeptide (or polypeptidefragment thereof) by substitution in said first nucleic acid sequence ofat least one different nucleic acid residue at a position thatcorresponds to one of the three nucleic acid residues in the codon thatencodes the amino acid corresponding to the amino acid at: 1) position65 of the hB7-1 polypeptide of SEQ ID NO:278, 2) position 56 of thehB7-2 polypeptide sequence at GenBank Acc. No. AAA58389; or 3) position50 of the hB7-2 polypeptide sequence at GenBank Acc. No. AAA86473. Themodified codon usually encodes an amino acid selected from the group ofHis, Arg, Lys, Pro, and/or Trp, and, typically, His. All codons encodingsuch amino acids are well known. Also provided are nucleic acidsequences (and fragments thereof that encode polypeptides having atleast one of the properties set forth above) that, but for codondegeneracy, hybridize under at least stringent or highly stringentconditions to one or more of the B7-1 or B7-2 nucleotide variants (orfragments thereof) described above, and nucleic acid sequencescomplementary to those described above.

Additional Aspects

The invention also includes a polypeptide comprising an ECD of an NCSMpolypeptide (or B7-1 or B7-2 polypeptide variant) of the invention withat least one of a signal peptide, transmembrane domain, and/orcytoplasmic domain. Usually, the transmembrane domain and/or cytoplasmicdomain is from a co-stimulatory molecule, such as an NCSM polypeptide ofthe invention or a B7-1 or B7-2 polypeptide. Any isolated or recombinantCD28BP or CTLA-4 polypeptide described above may further comprise atleast one of the following components: a signal sequence, transmembranedomain, or cytoplasmic domain. Various combinations of such from thevarious NCSM polypeptide sequences described herein can be made. In oneaspect, a signal sequence, transmembrane domain or cytoplasmic domainsignal sequence is selected from the signal sequence, transmembranedomain, or cytoplasmic domain, respectively, set forth in any of SEQ IDNOS:48–94, 174–252, 263–272, and 283–293.

A wide variety of signal peptide sequences can be fused to an ECD of aNCSM polypeptide or a B7-1 or B7-2 polypeptide variant to generate apolypeptide comprising at least a signal peptide sequence and an ECDpolypeptide. Signal peptides that can be used include, but are notlimited to, the amino acid sequence corresponding to a signal peptide ofa WT B7-1 or B7-2 polypeptide, including, but not limited to, amammalian or primate B7-1 or B7-2 polypeptide, and a signal peptidesequence from tissue plasminogen activator (TPA). The signal peptidesequence facilitates expression of the ECD polypeptide in vitro and invivo and is typically cleaved from the ECD.

In some embodiments, the signal peptide sequence comprises about 33,about 34, about 35, or about 36 amino acids. For the NCSM polypeptidesequences showing in the sequence listing, the signal peptide istypically about 34 amino acids in length. The ECD of an NCSMpolypeptide, such as a CD28BP or CTLA-4BP molecule, or a B7-1 or B7-2polypeptide variant of the invention typically begins with the firstamino acid residue following the last amino acid residue of the signalpeptide sequence. The predicted cleavage site of a signal peptide forany of the NCSM polypeptide sequences (and nucleic acid sequencesencoding such NCSM polypeptides) described herein can be predicted bymethods known to those skilled in the art; see, e.g., Nielsen et al.,Protein Eng'g 10:1–6 (1997), www.cbs.dtu.dk/services/SignalP/, andwww.cbs.dtu.dk/services/SignalP/bg_prediction.html.

One of ordinary skill in the art would understand that the predictedboundary between the signal peptide and ECD of an NCSM polypeptide (orB7-1 B7-2 polypeptide variant) of the invention can be determined fromthis alignment by comparing the ECD sequence of WT hB7-1 with thesequence of the NCSM molecule of interest. As shown in FIGS. 2A–2B,e.g., the signal peptide sequence for WT hB7-1 generally ends with thethree amino acid residues -cysteine-serine-glycine (-CSG). As shown byalignment with hB7-1, the signal peptide sequence for many CD28BPs ofthe invention also ends with the residues (-CSG or -SG) (see, e.g.,CD28BP-15 (SEQ ID NO:66)). The signal peptide sequence for manyCTLA-4BPs ends the residues -cysteine-serine-glycine (-CSG) (FIGS.3A–3B).

The ECD sequence of WT hB7-1 generally begins with the three amino acidresidues valine-isoleucine-histidine- (VIH-). The ECD of a CTLA-4BPusually begins with the same VIH- sequence, as shown in the alignment ofCTLA-4BP sequences with the hB7-1 sequence (FIGS. 3A–3B). The ECD of anNCSM polypeptide (or B7-1 or B7-2 polypeptide variant) may begin with ahydrophobic amino acid residue. For example, in some such aspects, theECD of a CD28BP begins with hydrophobic residue. In some aspects, theECD sequence of a CD28BP begins with the three amino acid residuesisoleucine-threonine-proline- (ITP-) (see, e.g., CD28BP-15 (SEQ IDNO:66)). In some embodiments, the ECD of a CD28BP begins with a prolineresidue; in some such embodiments, the ECD begins with the three aminoacid residues -PKS. In other embodiments, the ECD of some CD28BPs beginswith residues -IS (see, e.g., SEQ ID NOS: 49, 50, 65 and 191). The ECDsequence of many CTLA-4BPs generally begins with amino acid residuesvaline-isoleucine-histidine- (VIH-).

An ECD NCSM polypeptide (or B7-1 or B7-2 polypeptide variant) may befused to a signal peptide sequence from a WT B7-1 or WT B7-2polypeptide, such as a mammalian or primate B7-1 or B7-2 polypeptide.Alternatively, an ECD NCSM polypeptide (or B7-1 or B7-2 polypeptidevariant) is fused to an amino acid sequence corresponding to a signalpeptide of any NCSM polypeptide of the invention, including, e.g., thesignal peptide sequence of SEQ ID NOS:48–94, 174–252, 263–272, and278–293.

A nucleic acid sequence encoding an ECD NCSM polypeptide (or B7-1 orB7-2 polypeptide variant) may be fused to a nucleic acid sequenceencoding a signal peptide. The nucleic acid sequence encoding the signalpeptide comprises the nucleic acid sequence encoding a signal peptide ofa WT B7-1 or WT B7-2 polypeptide, such as a mammalian or primate B7-1 orB7-2 polypeptide. In another aspect, a nucleotide sequence encoding anECD of an NCSM polypeptide (or of a B7-1 or B7-2 polypeptide variant) isfused to a nucleic acid sequence encoding a signal peptide sequence,wherein said nucleic acid sequence comprises a signal peptide codingnucleotide sequence of any NCSM nucleic acid or WT B7-1 or B7-2,including, e.g., the signal peptide coding nucleotide sequence of any ofSEQ ID NOS:1–47, 95–173, 253–262, and 273–277. In a particularembodiment, the invention provides a nucleic acid sequence comprisingencoding a signal peptide and ECD comprising amino acid residues 1–244of SEQ ID NO:66. For one such embodiment, the nucleic acid comprisesnucleotide residues 1–732 of SEQ ID NO:19.

The predicted boundary between the ECD and TMD in the hB7-1 sequence isbetween amino acid residues 242 and 243, based on numbering of thefull-length WT hB7-1 sequence (FIG. 2G). The predicted boundary betweenthe ECD and TMD of an NCSM polypeptide (or B7-1 or B7-2 polypeptidevariant) can be determined by comparison of the position of the aminoacid residues at the predicted ECD/TM boundary of hB7-1 with amino acidresidues at the corresponding positions in the NCSM polypeptide ofinterest. In one embodiment, the predicted ECD/TM boundary of CD28BP-15(SEQ ID NO:66) is between amino acid residues 244 and 245, the ECD andTMD of CD28BP-15 comprise residues 35–244 and 245–268 of SEQ ID NO:66,respectively. In another embodiment, based on the numbering of aminoacid residues in the full-length hB7-1 sequence, the predicted ECD/TMboundary of CTLA-4BP 5x4-12c (SEQ ID NO:86) is between amino acidresidues 242 and 243, the ECD and TMD of CTLA-4BP 5x4-12c compriseresidues 35–242 and 243–263 of SEQ ID NO:86, respectively.

Alternatively, the predicted boundary between the ECD and TMD of an NCSMpolypeptide (or B7-1 or B7-2 polypeptide variant described herein) isdetermined using a software program known by those of ordinary skill inthe art that identifies one or more hydrophobic amino acid residueslikely to be present at the beginning of the TMD, after the ECD. Theprogram Vector NTI BioPlot analysis, version 6.0 Protein Scales viaProScales on ExPASy Server (Kyte J., Doolittle R. F., J. Mol. Biol.157:105–132(1982)) can be used, e.g., to determine hydropathicityregions of a polypeptide sequence and predict, e.g., the transmembraneregion of an NCSM polypeptide, B7-1 or B7-2 polypeptide variant of theinvention. With this program, the following parameters can be selected:Amino acid scale: Hydropathicity; window size: 9; relative weight of thewindow edges compared to the window center (in %): 100%; weightvariation model (if the relative weight at the edges is <100%): linear;and the scale need not be normalized from 0 to 1.

In one aspect, the predicted ECD/TM boundary of CD28BP-15 is betweenamino acid residues 255 and 256, and the ECD and TMD of CD28BP-15comprise residues 35–255 and 256–272 of a polypeptide selected from thegroup of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, and preferablySEQ ID NO:66. In another aspect, the TMD comprises at least about aminoacid residues 35–244, 35–243, 35–242, 35–255, 35–254, or 35–253 of apolypeptide selected from any of SEQ ID NOS:48–68, 174–221, 283–285, and290–293, and preferably SEQ ID NO:66. In one aspect, the CD amino acidsequence comprises a cytoplasmic domain of a polypeptide selected fromany of SEQ ID NOS:48–68, 174–221, 283–285, and 290–293, preferably SEQID NO:66.

One of skill would recognize, however, that the ECD of a CD28BP,CTLA-4BP, or B7-1 or B7-2 polypeptide variant may contain additional(e.g., one, two, or three) amino acid residues or fewer amino acidresidues (e.g., one, two or three residues) at the N-terminus and/orC-terminus and still maintain equivalent or comparable properties as ofthe molecule as described herein, and that the present inventionincludes such embodiments. Also included are polypeptides comprising asubsequence of SEQ ID NOS:49 and 50, wherein said subsequencecorresponds to an ECD (e.g., co-stimulatory ECD).

Any isolated or recombinant CD28BP or CTLA-4BP polypeptide describedabove may comprise a soluble extracellular domain of the respectivefull-length CD28BP or CTLA-4BP polypeptide or a fragment (e.g.,truncated ECD) or subsequence thereof. Any such CD28BP or CTLA-4BPpolypeptide may comprise a fusion protein comprising a CD28BP-ECD orCTLA-4BP-ECD or fragment thereof and at least one additional amino acidsequence, which may comprise at least one Ig polypeptide. The at leastone Ig polypeptide may comprise at least one human IgG polypeptidecomprising an Fc hinge, a CH2 domain, and a CH3 domain.

Any isolated or recombinant CD28BP or CTLA-4BP polypeptide describedabove may also comprise a polypeptide purification subsequence. Thepolypeptide purification subsequence is selected from, e.g., an epitopetag, a FLAG tag, a polyhistidine sequence, and a GST fusion.

In addition, isolated or recombinant CD28BP or CTLA-4BP polypeptidedescribed above or a B7-1 or B7-2 polypeptide variant may comprise amodified amino acid. The modified amino acid can be, e.g., aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid or sugar moiety, polymer, and an amino acidconjugated to an organic derivatizing agent. Methods for modifying oneor more amino acids of CD28BP, CTLA-4BP, and/or B7-1 and B7-2polypeptide variants, including methods of glycosylating and pegylatingamino acids are known in the art; additional methods are set forth inPCT Application No. PCT/DK01/00094 (Publ. No. WO 01/58935), whichpublished on Aug. 16, 2001. The invention also provides a compositioncomprising at least one polypeptide of any CD28BP and/or CTLA-4BPpolypeptide described above and an excipient or carrier. In one aspect,the composition comprises an isolated or recombinant NCSM polypeptidecomprising the amino acid sequence SEQ ID NOS:48–94, 174–252, 263–272,and 283–293, or a fragment thereof and a carrier or excipient. TheCD28BP fragment has a CD28/CTLA-4 binding affinity ratio about equal toor greater than the CD28/CTLA-4 binding affinity ratio of human B7-1and/or an ability to induce a T cell response about equal to or greaterthan that induce by hB7-1. The CTLA-4BP fragment has a CTLA-4/CD28binding affinity ratio about equal to or greater than that of h7-1. Thecomposition may be a pharmaceutical composition including apharmaceutically acceptable excipient or carrier. Exemplary andpreferred compositions and pharmaceutically acceptable excipients andcarriers are described below.

Consensus Sequences and Subsequences

The present invention also includes at least one NCSM polypeptideconsensus sequence derived from a comparison of two or more NCSMpolypeptide sequences described herein. For example, the presentinvention includes at least one CD28BP or CTLA-4BP polypeptide consensussequences derived from a comparison of, respectively, two or more CD28BPor CTLA-4BP polypeptide sequences described herein. A CD28BP polypeptideconsensus sequence as used herein refers to a nonnaturally-occurring orrecombinant polypeptide that predominantly includes those amino acidresidues that are common to all CD28BP polypeptides of the presentinvention described herein (e.g., full-length and ECD polypeptides andfragments having activities described herein) and that includes, at oneor more of those positions wherein there is no amino acid common to allsubtypes, an amino acid that predominantly occurs at that position andin no event includes any amino acid residue that is not extant in thatposition in at least one CD28BP of the invention. A CD28BP polypeptideconsensus sequence may have at least one property of a CD28BPpolypeptide as described herein (e.g., CD28/CTLA-4 binding affinityratio at least about equal to greater than that of hB7-1; and/or abilityto enhance an immune response, stimulate T cell proliferation oractivation).

A CTLA-4BP polypeptide consensus sequence refers to anonnaturally-occurring or recombinant polypeptide that predominantlyincludes those amino acid residues which are common to all CTLA-4BPpolypeptides of the present invention (e.g., full-length and ECDpolypeptides) and that includes, at one or more of those positionswherein there is no amino acid common to all subtypes, an amino acidthat predominantly occurs at that position and in no event includes anyamino acid residue that is not extant in that position in at least oneCTLA-4BP of the invention. A CTLA-4BP consensus polypeptide may have atleast one property of a CTLA-4BP polypeptide as described herein (e.g.,CTLA-4/CD28 binding affinity ratio at least about equal to greater thanthat of hB7-1; suppress an immune response, or inhibit T cellproliferation or activation).

An alignment of the amino acid sequence of the full-length parental WThB7-1 with each R1 and R2 CD28BP amino acid sequence is shown in FIGS.2A–2H. An alignment of the amino acid sequence of the full-lengthparental WT hB7-1 with each R1 and R2 CTLA-4BP amino acid sequence isshown in FIGS. 3A–3H. Both figures also show the regions of hB7-1corresponding to the signal peptide, ECD, transmembrane domain,cytoplasmic domain, and mature region (see arrows).) As shown, a numberof the CD28BP and CTLA-4BP sequences include two additional amino acidresidues in the putative signal sequence, as shown by comparison ofthese recombinant (chimeric) NCSMs with hB7-1; thus, the ECD for thesesequences putatively begins at amino acid residue 37. Of the 7 parentalspecies used for recursive sequence recombination, only the bovine aminoacid sequence includes two additional amino acid residues in theputative signal peptide sequence.

In one aspect, the invention provides the CD28BP consensus polypeptidesequence (SEQ ID NO:283) and the CTLA-4BP consensus polypeptide sequence(SEQ ID NO:286) and respective fragments or subsequences thereof thathave at least one property of a CD28BP or CTLA-4BP polypeptide asdescribed herein. A subsequence of a CD28BP or CTLA-4 consensus sequenceincludes a sequence that substantially corresponds (via visualinspection of alignment) to each of the ECD, transmembrane domain,cytoplasmic domain, signal peptide, or mature region of any respectiveCD28BP or CTLA-4BP polypeptide shown in the alignment in FIGS. 2A–2H and3A–3H.

The present invention also includes fragments and subsequences of theother CD28BP and CTLA-4BP amino acid sequences shown in FIGS. 2A–2H and3A–H, respectively, and nucleic acids encoding such fragments andsubsequences. Some such CD28BP and CTLA-4BP amino acid fragments andsubsequences have at least one property similar or equivalent (orimproved upon) to a CD28BP or CTLA-4BP polypeptide, respectively, asdescribed above.

In particular, the invention includes amino acid fragments orsubsequences of the CD28BP or CTLA-4BP shown in FIGS. 2A–2H and 3A–H,respectively, and nucleic acid sequences encoding such fragments andsubsequences, wherein said fragments or subsequences comprise at leastone of the mature domain, ECD, transmembrane domain, signal peptide,and/or cytoplasmic domain of the CD28BP or CTLA-4BP sequences shown inFIGS. 2A–2H and 3A–H. These domains may be identified by functionalanalysis, expression pattern, or comparison by amino acid (or nucleicacid) alignment with a corresponding domain of a WT B7-1 sequence.

For example, a hB7-1 polypeptide (or polynucleotide) sequence is alignedwith a fragment or subsequence of the invention, with amino acid (ornucleic acid) residues being aligned at equivalent positions. Thenumbering of amino acid residues (or nucleic acid residues) in aparticular domain, such as the ECD, for a CD28BP or CTLA-4BP fragment orsubsequence is based upon the numbering of residues in the correspondingCD28BP or CTLA-4BP polypeptide (or polynucleotide) sequence or, ifdesired, upon the amino acid numbering in a parental or WT B7-1polypeptide (or polynucleotide) sequence, such as hB7-1. The amino acidscomprising a signal sequence, ECD, mature domain, transmembrane domain,or cytoplasmic domain of a C28BP or CTLA-4BP polypeptide of theinvention, or polynucleotide encoding same, can be determined byalignment with a corresponding region of a WT B7-1 (e.g., hB7-1)polypeptide, or polynucleotide encoding the same; positions equivalentto those for the WT B7-1 (FIGS. 2A–2H and 3A–3H) can be determined.

The invention also provides at least one fragment of an isolated orrecombinant CD28BP polypeptide sequence selected from at least one ofSEQ ID NOS:48–68, 174–221, 283–285, and 289–293, wherein the fragmentbinds or specifically binds with a CD28 and/or CTLA-4 receptor and/orinduces T cell proliferation or activation in conjunction withstimulation of a T cell receptor (e.g, by antigen) as described hereinfor CD28BP polypeptides, and provided the fragment itself is not anamino acid fragment known in the art to have such properties.

In addition, the invention provides at least one fragment of an isolatedor recombinant CTLA-4BP polypeptide sequence selected from at least oneof SEQ ID NOS:69–92, 222–272, and 286–288, wherein the fragment binds orspecifically binds with a CD28 and/or CTLA-4 receptor and/or inhibits Tcell activation or proliferation as described herein for CTLA-4BPpolypeptides, and further provided the fragment itself is not an aminoacid fragment known in the art to have such properties. Fragments of SEQID NOS:93–94 having such properties as described for either of CTLA-4BPor CD28 polypeptides are also included.

Also provided are polypeptide sequences corresponding to at least one ofthe following components of any of SEQ ID NOS:48–94, 174–252, 263–272,and/or 283–293: a signal peptide, ECD, transmembrane domain, cytoplasmicdomain, or mature region or any combination thereof of such components,such as, e.g., a signal peptide and ECD. A recombinant polypeptidecomprising one or more of any of these individual components from onesuch sequence fused to one or more of these individual components atleast one additional sequence is also contemplated in the invention.

Making Polypeptides

Recombinant methods for producing and isolating NCSM polypeptides of theinvention are described above. In addition to recombinant production,the polypeptides may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, W H Freeman Co, San Francisco; Merrifield J. (1963) JAm Chem Soc 85:2149–2154). Peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to providefull-length NCSM polypeptides or fragments thereof. Alternatively, suchsequences may be ordered from any number of companies which specializein production of polypeptides. Most commonly, NCSM polypeptides areproduced by expressing coding nucleic acids and recovering polypeptides,e.g., as described above.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any NCSM nucleic acid described herein, which is operativelylinked to a regulatory sequence effective to produce the encodedpolypeptide, culturing the cells in a culture medium to produce thepolypeptide, and isolating the polypeptide from the cells or from theculture medium. An amount of nucleic acid sufficient to facilitateuptake by the cells (transfection) and/or expression of the NCSMpolypeptide is utilized. The culture medium can be any described hereinand in the Examples. The nucleic acid is introduced into such cells byany delivery method described herein, including, e.g., injection, genegun, passive uptake, etc. The NCSM nucleic acid may be part of a vector,such as a recombinant expression vector, including a DNA plasmid vector,or any vector described herein. The nucleic acid or vector comprising aNCSM nucleic acid may be prepared and formulated as described herein,above, and in the Examples below. Such a nucleic acid or expressionvector may be introduced into a population of cells of a mammal in vivo,or selected cells of the mammal (e.g., tumor cells) may be removed fromthe mammal and the nucleic acid expression vector introduced ex vivointo the population of such cells in an amount sufficient such thatuptake and expression of the encoded polypeptide results. Or, a nucleicacid or vector comprising a NCSM nucleic acid is produced using culturedcells in vitro. In one aspect, the method of producing a NCSMpolypeptide comprises introducing into a population of cells arecombinant expression vector comprising any NCSM nucleic acid describedherein in an amount and formula such that uptake of the vector andexpression of the NCSM polypeptide will result; administering theexpression vector into a mammal by any introduction/delivery formatdescribed herein; and isolating the polypeptide from the mammal or froma byproduct of the mammal.

Using Polypeptides

-   -   Antibodies

In another aspect of the invention, a NCSM polypeptide or fragmentsthereof of the invention is used to produce antibodies which have, e.g.,diagnostic, therapeutic, or prophylactic uses, e.g., related to theactivity, distribution, and expression of NCSM polypeptides andfragments thereof. Antibodies to NCSM polypeptides or peptide fragmentsthereof of the invention may be generated by methods well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, humanized, single chain, Fab fragments andfragments produced by a Fab expression library. Antibodies, e.g., thosethat block receptor binding, are especially preferred for therapeuticand/or prophylactic use.

NCSM polypeptides for antibody induction do not require biologicalactivity; however, the polypeptides or oligopeptides are antigenic.Peptides used to induce specific antibodies may have an amino acidsequence consisting of at least about 10 amino acids, preferably atleast about 15 or 20 amino acids or at least about 25 or 30 amino acids.Short stretches of a NCSM polypeptide may be fused with another protein,such as keyhole limpet hemocyanin, and antibody produced against thechimeric molecule.

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art, and many antibodies are available. See, e.g.,Current Protocols in Immunology, John Colligan et al., eds., Vols. I–IV(John Wiley & Sons, Inc., NY, 1991 and 2001 Supplement); and Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;and Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)Nature 256:495–497. Other suitable techniques for antibody preparationinclude selection of libraries of recombinant antibodies in phage orsimilar vectors. See, Huse et al. (1989) Science 246:1275–1281; and Wardet al. (1989) Nature 341:544–546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better.

Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed.) (1995) Antibody Engineering, 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,N.J. (Paul).In one useful embodiment, this invention provides for fullyhumanized antibodies against the NCSM polypeptides of the invention orfragments thereof. Humanized antibodies are especially desirable inapplications where the antibodies are used as therapeutics and/orprophylactics in vivo in human patients. Human antibodies consist ofcharacteristically human immunoglobulin sequences. The human antibodiesof this invention can be produced in using a wide variety of methods(see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, and BorrebaeckMcCafferty and Paul, supra, for a review). In one embodiment, the humanantibodies of the present invention are produced initially in triomacells. Genes encoding the antibodies are then cloned and expressed inother cells, such as nonhuman mammalian cells. The general approach forproducing human antibodies by trioma technology is described by Ostberget al. (1983), Hybridoma 2:361–367, Ostberg, U.S. Pat. No. 4,634,664,and Engelman et al., U.S. Pat. No. 4,634,666. The antibody-producingcell lines obtained by this method are called triomas because they aredescended from three cells—two human and one mouse. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells.

Sequence Variations

-   -   Conservatively Modified Variations

NCSM polypeptides of the present invention include conservativelymodified variations of the sequences of any of SEQ ID NOS:48–94,174–252, 263–272, and 283–293 and fragments thereof. Such conservativelymodified variations comprise substitutions, additions or deletions thatalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than about 5%, more typically less than about 4%,2%, or 1%) in any of SEQ ID NOS:48–94, 174–252, 263–272, and 283–293.

For example, a conservatively modified variation (e.g., a deletion) ofthe 296 amino acid polypeptide identified herein as SEQ ID NO:48 willhave a length of about 282 amino acids, preferably about 285 aminoacids, more preferably about 288 amino acids, still more preferablyabout 291 amino acids, and still even more preferably about 294 aminoacids or more, corresponding to a deletion of less than about 5%, 4%,3%, 2%, or 1% of the polypeptide sequence.

Another example of a conservatively modified variation (e.g., a“conservatively substituted variation”) of the polypeptide identifiedherein as SEQ ID NO:48 will contain “conservative substitutions,”according to the six substitution groups set forth in Table 2 (supra),in up to about 15 residues (i.e., less than about 5%) of the 296 aminoacid polypeptide.

As an example, if four conservative substitutions were localized in theregion corresponding to amino acids 69–94 of SEQ ID NO:48, examples ofconservatively substituted variations of this region,

QKDSK MVLAI LPGKV QVWPE YKNRTI, would include:

NKDSK MVVAI LPGKV QVFPE YKNKTI and

QKDAK MVLAI LPGRV QMWPE YKQRTI and the like, where conservativesubstitutions are underlined.

The NCSM polypeptide sequences of the invention or fragments thereof,including conservatively substituted sequences, can be present as partof larger polypeptide sequences such as occur upon the addition of oneor more domains for purification of the protein (e.g., poly-hissegments, FLAG tag segments, etc.). These additional functional domainseither have little or no effect on the activity of the NCSM portion ofthe protein, or the additional domains can be removed by post synthesisprocessing steps such as by treatment with a protease, inclusion of anintein, or the like.

Defining NCSM Polypeptides by Immunoreactivity

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with a polypeptideencoded by the second nucleic acid. A polypeptide is typicallysubstantially identical to a second polypeptide, e.g., where the twopeptides differ only by conservative substitutions.

The phrase “specifically (or selectively) binds,” “specifically (orselectively bound,” or “specifically (or selectively) immunoreactivewith,” when referring to a polypeptide, refers to a binding reactionwith an antibody which is determinative of the presence of thepolypeptide, or an epitope from the polypeptide, in the presence of aheterogeneous population of polypeptides and other biologics. Specificbinding between an antibody or other binding agent and an antigengenerally means a binding affinity of at least about 10⁵ to 10⁶ M⁻¹.

Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular polypeptide and do not bind in a significant amountto other polypeptides present in the sample. The antibodies raisedagainst a multivalent antigenic polypeptide will generally bind to thepolypeptides from which one or more of the epitopes were obtained.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particularpolypeptide. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular polypeptide.For example, solid-phase ELISA immunoassays, Western blots, orimmunohistochemistry are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (hereinafter “Harlow and Lane”), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Typically, a specific or selective reaction is atleast twice background signal or noise and more typically 2.5×–5× ormore than 10 to 100 times background.

The polypeptides of the invention provide structural features that canbe recognized, e.g., in immunological assays. The generation of antiseracontaining antibodies (for at least one antigen) which specificallybinds the polypeptides of the invention, as well as the polypeptideswhich are bound by such antisera, are a feature of the invention.Preferred binding agents, including antibodies described herein, bindNCSM polypeptides and fragments thereof with affinities of at leastabout 10⁶ to 10⁷ M⁻¹, and preferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹.Conventional hybridoma technology can be used to produce antibodieshaving affinities of up to about 10⁹ M−1. However, new technologies,including phage display and transgenic mice, can be used to achievehigher affinities (e.g., up to at least about 10¹² M⁻¹). In general, ahigher binding affinity is advantageous.

The invention includes NCSM polypeptides and fragments thereof thatspecifically bind to or that are specifically immunoreactive with anantibody or polyclonal antisera generated against at least one immunogencomprising at least one amino acid sequence selected from one or more ofSEQ ID NOS:48–94, 174–252, 263–272, and 283–293 or fragments thereof. Toeliminate cross-reactivity with other peptides, the antibody or antiserais subtracted with polypeptides encoded by sequences such as, e.g.,those represented at GenBank accession numbers A92749, A92750, AA983817,AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894,AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829,AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443,AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843,L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191,S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832,U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. Where theGenBank sequence corresponds to a nucleic acid, a polypeptide encoded bythe nucleic acid is generated and used for antibody/antisera subtractionpurposes. Where the nucleic acid corresponds to a non-coding sequence,e.g., a pseudo-gene, an amino acid which corresponds to the readingframe of the nucleic acid is generated (e.g., synthetically), or isminimally modified, e.g., to include a start codon, promoter or the likefor recombinant production.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more NCSM polypeptides comprising one or moreof the sequences corresponding to one or more of SEQ ID NOS:48–94,174–252, 263–272, and 283–293, or a substantial subsequence or fragmentthereof (i.e., comprising at least about 30%, 40%, 50%, 60%, 70%, 80%,90% or more of the amino acids of the full length sequence provided).The full set of potential polypeptide immunogens derived from SEQ IDNOS:48–94, 174–252, 263–272, and 283–293 are collectively referred toherein as “the immunogenic polypeptides.” The resulting antisera isoptionally selected to have low cross-reactivity against the control,e.g., co-stimulatory homologues and any such cross-reactivity is removedby immunoabsorption with one or more of the control polypeptides, priorto use of the polyclonal antiserum in the immunoassay. Sequences whichare substantially identical to such sequences can also be used, e.g.,which are at least about 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical, e.g., asdetermined using BLAST or the other algorithms described herein andabove, e.g., using default parameters.

In another aspect, the invention provides an antibody or antiseraproduced by administering a NCSM polypeptide of the invention to amammal, which antibody or antisera specifically binds one or moreantigens, the one or more antigens comprising a polypeptide comprisingone or more of the amino acid sequences SEQ ID NOS:48–94, 174–252,263–272, and 283–293, which antibody or antisera does not specificallybind to a polypeptide encoded by one or more of GenBank NucleotideAccession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651,AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519,AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832,AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645,AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073,M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696,U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622,X60958, Y08823, and Y09950.

The antisera comprises the serum of a subject that has been immunizedagainst at least one antigen. The antiserum can be monovalent orpolyvalent; that is, it can contain antibodies specific for one or moreantigenic determinants, depending on whether the subject was immunizedwith one antigen or a mixture of antigens.

Also included is an antibody or antisera (comprising one or moreantibodies) which specifically binds a polypeptide comprising a sequenceselected from: SEQ ID NOS:48–94, 174–252, 263–272, and 283–293, whereinthe antibody or antisera (comprising antibodies) does not specificallybind to a polypeptide encoded by one or more of GenBank NucleotideAccession Nos. set forth above.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein may be produced in a mammaliancell line. An inbred strain of mice (used in this assay because resultsare more reproducible due to the virtual genetic identity of the mice)is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see Harlow and Lane, supra, for a standarddescription of antibody generation, immunoassay formats and conditionsthat can be used to determine specific immunoreactivity). Alternatively,one or more synthetic or recombinant polypeptides derived from thesequences disclosed herein is conjugated to a carrier protein and usedas an immunogen.

Polyclonal antisera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control co-stimulatorypolypeptides to produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control polypeptides. Preferably at least two ofthe immunogenic NCSM polypeptides are used in this determination,preferably in conjunction with at least two of the control polypeptides,to identify antibodies which are specifically bound by the immunogenicpolypeptide(s).

In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5–10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic NCSM molecules ascompared to binding to any control polypeptides. That is, the stringencyof the binding reaction is adjusted by the addition of non-specificcompetitors such as albumin or non-fat dry milk, or by adjusting saltconditions, temperature, or the like. These binding conditions are usedin subsequent assays for determining whether a test polypeptide isspecifically bound by the pooled subtracted polyclonal antisera. Inparticular, test polypeptides which show at least a 2–5× higher signalto noise ratio than the control polypeptides under discriminatorybinding conditions, and at least about a ½ signal to noise ratio ascompared to the immunogenic polypeptide(s), share substantial structuralsimilarity with the immunogenic polypeptides as compared relative toknown B7-1 or related co-stimulatory polypeptides, and are thus NCSMpolypeptides of the invention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorption with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is determined as compared tothe ability of the immunogenic polypeptide(s) to compete for binding tothe antisera. Again, the percent cross-reactivity for the controlpolypeptides is calculated, using standard calculations. Where thepercent cross-reactivity is at least 5–10× as high for the testpolypeptides, the test polypeptides are said to specifically bind thepooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic polypeptide(s). In order to make thiscomparison, the two polypeptides are each assayed at a wide range ofconcentrations and the amount of each polypeptide required to inhibit50% of the binding of the subtracted antisera to the immobilized proteinis determined using standard techniques. If the amount of the testpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the test polypeptide is said tospecifically bind to an antibody generated to the immunogenic protein,provided the amount is at least about 5–10× as high as for a controlpolypeptide.

As a final determination of specificity, the pooled antisera isoptionally fully immunoabsorbed with the immunogenic polypeptide(s)(rather than any control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunoabsorbtion is detectable.This fully immunoabsorbed antisera is then tested for reactivity withthe test polypeptide. If little or no reactivity is observed (i.e., nomore than 2× the signal to noise ratio observed for binding of the fullyimmunoabsorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Proliferation/Activation and Anti-Proliferation/Inactivation Propertiesof NCSM Molecules

The effect of the NCSM polypeptides and fragments thereof was examinedon T cells as described in the Examples, infra. The results indicatethat compositions comprising a NCSM polypeptide of the present inventionor fragment thereof, or a soluble NSCM-ECD and NCSM-ECD-Ig, can be usedin methods of the invention to induce or inhibit proliferation and/oractivation of T cells, for example, in conjunction with stimulation of Tcell receptor (e.g., by antigen or anti-CD3 Ab). The ability of a NCSMpolypeptide or fragment thereof to induce or inhibit T cellproliferation/activation is typically measured against the ability ofwild-type B7-1 (such as, e.g., a human, primate, or cow B7-1) to induceor inhibit T cell proliferation or activation, e.g., in conjunction withstimulation of T cell receptor (e.g., by antigen or anti-CD3 Ab).Similarly, a NCSM polynucleotide that encodes such a NCSM polypeptide orfragment thereof, or a soluble NSCM-ECD and NCSM-ECD-Ig, can be used inmethods of the invention to induce or inhibit proliferation and/oractivation of T cells by using e.g., cells transfected with andexpressing or secreting such NCSM molecules. Here, too, the ability ofthe expressed or secreted NCSM peptide molecule to induce or inhibit Tcell proliferation and/or activation is measured in the same manner.

Inducing or inhibiting of proliferation/activation of T cells can beperformed in vitro (as useful, e.g., in a variety of proliferationassays or in generation of, e.g., tumor-antigen specific T cells thatcan be administered to cancer patients), or in vivo (as useful, e.g., asa therapeutic and/or prophylactic).

CD28BPs and CTLA-4BPs of the invention (and fragments thereof) and thenucleic acids encoding such CD28BPs and CTLA-4BPs of the invention (andfragments thereof) are useful in numerous applications, either when usedas gene-based therapeutics/vaccines or when administered as, e.g.,soluble polypeptides, proteins, or fragments thereof, in the presence orabsence of a specific antigen or mixture of antigens.

Compositions of the present invention can be used to prophylactically ortherapeutically treat and thereby prevent, alleviate or ameliorate avariety of conditions where stimulation of T cellproliferation/activation or inhibition of T cell proliferation and/oractivation would be beneficial to a patient. Such uses include, but arenot limited to, e.g., prophylaxis of infectious disease, therapeutic andprophylactic treatment of a variety of chronic infectious diseases,cancers, allergies, autoimmune diseases, septic shock, prevention andtreatment of graft versus host disease, and the like; and the preventionof organ transplant rejection and the like.

The products of the invention can also be used in gene therapy to reduceimmune system recognition of cells expressing a transgene, thusprolonging the longevity of the expression of the transgene. Generationof transgenic animals expressing products of the invention optionallycan be used as sources of organs for humans (e.g., the organs express aCTLA-4BP of the invention which, on the surface of the organ,down-regulates host T cell responses thus reducing risk of rejection),etc.

A desired goal in developing CTLA-4BPs was to create NCSM molecules thatspecifically signal through CTLA-4 and thus can, e.g., induce tolerance,suppress activated T cells, and induce regulatory T cells. Other routesof modifying immune responses (e.g., nonspecific immunosuppression withcyclosporin A, blockage of APC—T cell interaction with CTLA-4-Ig or withAnti-B7 monoclonal antibodies (mAbs), or blockage of APC—B-cellactivation with Anti-CD40L Abs) have drawbacks (e.g., they inducegeneral immunosuppression or they inhibit T cell growth, etc.) and donot achieve the goal of CTLA-4BPs of the invention, namely induction oftolerance of antigen-specific T cells. An example (but not limiting)application of CTLA-4BP in gene therapy is illustrated by, e.g., using avector encoding a CTLA-4BP and a transgene (alternatively the CTLA-4BPand transgene can be on separate plasmids) which is introduced into atarget cell and whose gene products are presented on the same cellsurface. The transgene (in context with MCH) interacts with the T cellreceptor (TCR) on the T cell while the CTLA-4BP interacts with CTLA-4 onthe T cell, all of which leads to an inhibition or reduction of T cellresponse and a prolonged expression of the transgene.

The products (i.e., polypeptides, nucleic acids, and fragments thereof)of the present invention can be useful in such things as vaccineadjuvants (e.g., for genetic vaccines, protein vaccines, attenuated orkilled viral vaccines). For example, the nucleic acids of CD28BPs andCTLA-4BPs (or fragments thereof) can be components of genetic vaccinesand gene therapy vectors (e.g., DNA vaccines, viral vectors), or theycan be expressed in cells of interest (e.g., tumor cells, dendriticcells) which then can be used as vaccines or therapeutics. Additionally,products of the invention can be transfected into tumor cells which,after being rendered unable to proliferate (e.g., by irradiation) thencan be used as cell-based vaccines. Alternatively, such transfectedcells are lysed and the resulting lysate used as a vaccine. CD28BPs canserve as T cell adjuvants and administered in either as nucleic acids(including, e.g., vectors comprising nucleic acids encoding CD28BPs) oras proteins. Use of a wild-type human B7 gene as a component in a DNAvaccine along with an antigen(s) of interest results in both positiveand negative signals to T cells since wild-type human B7 can bind withboth CD28 and CTLA-4 on T cells. However, DNA vaccines encoding aproduct of the invention, e.g., CD28BP or fragments thereof, canselectively tailor the T cell response, e.g., CD28BP in a DNA vaccinewill result in positive signals to T cells (e.g., signals to induce Tcell proliferation/activation). For example, a CD28BP is optionally usedin a treatment vaccine for melanoma (in the context of TRP-1, TRP-2and/or tyrosinase). An illustrative, but not limiting example is:intradermal injection of DNA (antigen) followed by subcutaneousinjection of protein (CD28BP) with time periods of, e.g., 2 weeksbetween treatments for, e.g., 4 cycles. The CD28BP presents low risk ofcross-reactivity with wild-type in treatment of life threateningmelanoma. Fragments of the CD28BP encoding nucleic acid are optionallyused in the procedure. As another illustrative, but not limitingexample, CTLA-4BPs of the invention or fragments thereof are optionallyused in conjunction with MBP (myelin basic protein) in a treatmentvaccine for multiple sclerosis which is given, e.g., as an intramuscularinjection every 3 weeks for 6 months or as the condition warrants.

A gene-based vaccine utilizing a NCSM (e.g., a CD28BP or CTLA-4BP) orfragments thereof is optionally comprised of a plasmid encoding both theantigen(s) of interest and the NCSM (e.g., either CD28BP or CTLA-4BP)(alternatively, the antigen(s) of interest is on a separate plasmid fromthe NCSM gene (e.g., the CD28BP or CTLA-4BP gene(s)). The products ofthe genes of the plasmid(s) are expressed on the surface of, e.g., anAPC. Interaction occurs between the antigen of interest (in the contextof MHC) and CD28BP (both on the, e.g., APC) with, respectively, the Tcell receptor and CD28 on the T cell which leads to T cellproliferation/activation. Optionally, interaction occurs between theantigen of interest (in the context of MHC) and CTLA-4BP (both on the,e.g., APC) with, respectively, the TCR and CTLA-4 on the T cell whichleads to T cell anergy/tolerance.

Another example of CD28BP application is illustrated by inducingspecific T cell activation through use of a plasmid encoding a CD28BP orfragments thereof. The plasmid is transfected into a tumor cell (e.g.,ex vivo), which is, e.g., irradiated to stop proliferation, and is thenused as a vaccine (or optionally a tumor cell lysate, e.g., Melacine® isused). The tumor antigens are presented (in context with MHC) to the Tcell and interact with the TCR. Additionally, the CD28BP expressed onthe cell along with the tumor antigen is presented to the T cell andinteracts with CD28, thus leading to T cell activation.

Soluble NCSM Polypeptides and Nucleic Acids

The present invention provides soluble NCSM polypeptides (or fragmentsthereof) and nucleic acids encoding them. Selected regions (e.g., theECD, truncated extracellular domain, secreted subsequence of a NCSMpolypeptide) or fragments thereof are provided in both polypeptide andnucleic acid format. These soluble molecules are suited for use asprophylactics, therapeutics, and/or diagnostic tools and can be targetedor designed for specific actions and a variety of applications asdescribed herein.

Soluble B7-1 proteins and fragments have been described andcharacterized. See, e.g., U.S. Pat. No. 6,071,716. Standard proceduresfor expressing soluble B7-1 proteins and fragments thereof, recoveringsuch molecules from culture media, screening and characterizing suchmolecules for e.g., T cell proliferation or lymphokine production, asdescribed in, e.g., U.S. Pat. No. 6,071,716, can be used and applied tosoluble NCSM polypeptides and fragments thereof of the presentinvention.

A “soluble” NCSM polypeptide, such as a soluble CD28BP or CTLA-4BP ofthe invention, means a polypeptide comprising an amino acid sequencethat corresponds to that of the extracellular domain (ECD) of a NCSMpolypeptide or a fragment of said ECD (e.g., a truncated ECD). Thesoluble NCSM polypeptide typically does not include the amino acidsequences corresponding to the full-length cytoplasmic or transmembranedomain. The amino acid sequence corresponding to the signal peptide orleader, or a fragment thereof, may or may not be included in the solubleNCSM polypeptide. A soluble NSCM polypeptide may further comprise animmunoglobulin (Ig) or Ig fragment, such as, e.g., an Fc portion of anIg (e.g., IgG) linked to an NCSM ECD or fragment thereof. In one aspect,a soluble NCSM polypeptide comprises a fusion protein comprising an NCSMECD or fragment thereof and an Ig or fragment thereof, including, e.g.,an Fc portion. The Ig may be from a human, primate, or other mammal. Asoluble NCSM polypeptide is freely secreted into the medium surroundinga host cell when it is recombinantly produced in the host cell. Nucleicacids encoding any such soluble NCSM polypeptides (or fragments thereof)described above and hereinafter are also an aspect of the invention.

For each NCSM molecule of the invention, a putative ECD polypeptidesequence (or nucleotide sequence encoding said polypeptide sequence) maybe determined by alignment of the NCSM ECD polypeptide sequence (ornucleotide sequence encoding same) with an analogous ECD polypeptidesequence (or nucleotide sequence encoding same) of human B7-1 or othermammalian B7-1 (e.g., primate). The putative amino acid and nucleic acidsequences corresponding to the respective putative signal peptide,transmembrane domain, cytoplasmic domain, and mature region can also besimilarly determined for each NCSM molecule of the invention. It isreadily understood by one of ordinary in the art that each of thesedomains/regions of the NCSM polypeptides and polynucleotides determinedby such alignment comparison is putative and thus may vary in length byone or more amino acids or nucleic acids, respectively. One of skill canreadily confirm such domains/regions by other analyses known in the art,including those used to determine corresponding domains/regions inhB7-1.

The soluble NCSM molecules can show preferential binding to eitherCTLA-4 or CD28 receptor. Soluble CD28BPs (and fragments thereof) canbind preferentially with CD28 and CTLA-4BPs (and fragments thereof) canbind preferentially with CTLA-4 as compared to the binding of solublewild-type (WT) human B7-1 to CD28 and CTLA-4. For example, when anantigen is presented (in context with MHC) on the surface of a cellwhere it interacts with the TCR on a T cell, a soluble CD28BP optionallycan interact simultaneously with the T cell through the CD28 molecule,thus leading to T cell proliferation/activation. Conversely, when anantigen is presented (in context with MHC) on the surface of a cellwhere it interacts with the TCR on a T cell while simultaneously asoluble CTLA-4BP also interacts with the T cell (through the CTLA-4molecule), T cell anergy/tolerance can result.

Optionally, and additionally, the soluble NCSM molecules of theinvention, or fragments thereof, can be used as agonists or antagonistsof the respective T cell receptors. The soluble CD28BP molecules canoptionally act as agonists by stimulating T cellproliferation/activation by binding with CD28 or the soluble CD28BPmolecules can optionally act as antagonists by binding with CD28 withoutstimulating T cell proliferation/activation. Furthermore, solubleCTLA-4BP molecules can optionally act as agonists by binding with CTLA-4and inhibiting T cell proliferation/activation (e.g., not stimulating Tcells) or the soluble CTLA-4BP molecules can act as antagonists bybinding with CTLA-4 and not inhibiting T cell proliferation/activation.

These soluble NCSM molecules can be delivered to a subject by a varietyof formats. For example, the soluble NCSM molecules can be delivered aspolypeptides or proteins (or fragments thereof) or as nucleic acids (orfragments thereof) encoding such polypeptides or proteins.

In one aspect, the invention includes an isolated or recombinant nucleicacid comprising a polynucleotide sequence selected from: (a) apolynucleotide sequence selected from SEQ ID NOS:1–21 and 95–142, or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence encoding a polypeptide selected from SEQ ID NOS:48–68, 174–221,283–285, and 290–293, or a complementary polynucleotide sequencethereof; (c) a polynucleotide sequence which, but for the degeneracy ofthe genetic code, hybridizes under at least stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b);and (d) a polynucleotide sequence comprising all or a nucleotidefragment of (a), (b), or (c), wherein the nucleotide fragment encodes asoluble polypeptide having a CD28/CTLA-4 binding affinity ratio aboutequal to or greater than the CD28/CTLA-4 binding affinity ratio of humanB7-1 and/or a soluble polypeptide that, in the presence of activated Tcells, down-regulates or inhibits a T cell proliferation responsecompared to the response of soluble hB7-1 (e.g., hB7-1-ECD orhB7-1-ECD-Ig)—that is causes a lower T cell proliferation response thatdoes soluble hB7-1.The present invention also provides fusion proteins,including soluble fusion proteins, comprising a fusion of a protein(such as a CD28BP, CTLA-4BP, B7-1 polypeptide variant, or B7-2polypeptide variant of the current invention) or fragments thereof withan immunoglobulin (Ig) or portion of an immunoglobulin. The resultingprotein-Ig fusions (or immunoadhesions or Fc-fusion proteins) can showimproved pharmacokinetics, such as longer half-life in vivo, and/orincreased expression. Such fusion proteins can also simplifypurification and augment isotype effector functions for specificproteins. See, e.g., Ashkenazi, A. et al. (1997) Curr Op in Immunol9(2): 195–200 for a review of the uses and applications of Ig-proteinfusions, which is incorporated by reference in its entirety for allpurposes. Examples of Ig protein fusion components, including peptidelinkers and Fc regions, that are can be fused to NCSM molecules of theinvention, including full-length NCSM molecules and at least onecomponent thereof (e.g., signal peptide, ECD, TM, and/or CD of suchNCSM), and B7-1 and B7-2 variants of the invention, are provided inAshkenazi et al. For example, the invention includes a nucleic acidsequence encoding the signal peptide and ECD of an NCSM described hereincan be fused to a nucleotide sequence encoding a linker peptide and/orFc region and a fusion protein encoded therefrom. The NCSMs of theinvention can be fused to many variations of, e.g., immunoglobulins andthe NCSM fusions are not limited by, e.g., the type of Ig molecule used.In addition to full length NCSM molecules, fragments of NCSM molecules(e.g., the extracellular domain (ECD) or fragments of the ECD) can befused to Ig molecules. Various sequences, e.g., peptide linkersequences, proteolytic cleavage sites (such as, e.g., Factor Xa cleavagesite), etc., can also be incorporated into the NCSM-Ig fusion protein.In some embodiments, the small peptide linker forming the in-frametranslational coupling between an NCSM ECD (or hB7-1) and the IgG1 Fccomprised the amino acid sequence valine-threonine (VT) orglycine-valine-threonine (GVT), depending upon the nucleotide sequencecompatibility of the 3′ codon of the NCSM ECD (see, e.g., FIG. 14B andExample IV below). The length and nature of the amino acid sequence ofthe peptide linker positioned between the Fc region and ECD is believedimportant for providing proper contact between the Fc regions ofNCSM-ECD monomers or NCSM-ECD-Ig fusion monomers and facilitating orenhancing multimerization of such NCSM-ECD or NCSM-ECD-Ig monomers. Inone aspect, e.g., the linker sequence may comprise from at least aboutone to at least about 11 amino acids; the specific amino acid mayinclude residues GVT, as described above, or other amino acid residueshaving characteristics that facilitate aggregation of NCSM-ECD orNCSM-ECD-Ig fusion monomers. In another aspect, the invention providesmultimers of NCSM-ECD and NCSM-ECD-Ig fusion monomers. Nucleotidesequences encoding all such NCSM-ECCD monomers, and NCSM-Ig andNCSM-ECD-Ig fusion proteins are another aspect of the invention.

A fusion protein comprising a NCSM polypeptide or fragment thereof fusedto human IgG Fc domain is an aspect of the present invention, see, e.g.,Example 4, infra. The NCSM-Ig fusion proteins have the benefits of beingsoluble (thus, e.g., expanding their uses as prophylactics andtherapeutics) and being stabilized by the Ig portion of the fusion. Thesoluble NCSM polypeptide-IgG fusion proteins of the present inventionare suited for use as prophylactics and/or therapeutics since they canbe targeted or tailored for specific actions and a variety ofapplications.

The NCSM-Ig fusion proteins and polypeptides of the invention can beused for, e.g., similar applications (including, e.g., therapeutic,prophylactic, and diagnostic applications described herein) as the NCSMproteins and polypeptides of the invention (as indicated throughout).These Ig-fusion proteins of the invention also show preferential bindingto either CTLA-4 or CD28. CD28BP-Ig fusions (and fragments thereof) bindpreferentially with CD28 and CTLA-4BP-Ig fusions (and fragments thereof)bind preferentially with CTLA-4 as compared to the binding of human B7-1to CD28 and CTLA-4. For example, when an antigen is presented (incontext with MHC) on the surface of a cell where it interacts with theTCR on a T cell, a soluble CD28BP-Ig fusion protein optionally caninteract simultaneously with the T cell through the CD28 molecule, thusleading to T cell proliferation/activation. Conversely, when an antigenis presented (in context with MHC) on the surface of a cell where itinteracts with the TCR on a T cell while simultaneously a solubleCTLA-4BP-Ig fusion protein also interacts with the T cell (through theCTLA-4 molecule), T cell anergy/tolerance can result.

Optionally, the soluble NCSM-Ig fusion proteins (e.g., CD28BP-Ig andCTLA-4BP-Ig), or fragments thereof, or proteins (or protein fusions)based on the NCSMs (e.g., CD28BPs or CTLA-4BPs) are used as agonists orantagonists of the respective receptors on the T cell. For example,CD28BP or CD28BP-Ig fusions acting as agonists by stimulating T cellproliferation/activation through binding to CD28 or as antagonists bybinding to CD28 without inducing T cell proliferation/activation.Alternatively, CTLA-4 or CTLA-4BP-Ig fusions acting as agonists byinhibiting (or not stimulating) T cell proliferation/activation throughbinding to CTLA-4 or as antagonists by binding to CTLA-4 withoutinhibiting T cell proliferation/activation.

In one aspect, the IgG Fc hinge, CH2 and CH3 domains are modified orevolved using a recursive sequence recombination method, such as DNAshuffling, or another diversity generation method to produce a libraryof recombinant IgG Fc hinge CH2 and CH3 domains from a group of selectedparental IgG Fc sequences, and then screening the library by appropriatescreening procedures to identity a chimeric IgG Fc comprising at leastone recombined Fc constant domain exhibiting reduced binding to at leastone Fcγ receptor—e.g., FcγRII and FcγRIII. Specifically, the library ofrecombinant (shuffled) IgG molecules are digested with BsteII and EcoRI,and ligated into a BstEII—EcoRI digested plasmid comprising at least oneNCSM nucleic acid sequence to produce a non- or reduced-FcγR bindingNCSM-Ig fusion protein molecules. Supernatants from 293 cellstransiently transfected with such expression plasmids are incubated with293 cells expressing either FcγRII or FcγRIII, and binding affinity ofthe expressed recombinant clones is determined by FACS analysis methodsusing a NCSM-specific mAb conjugated with FITC. IgG Fc variantsexhibiting reduced or no binding to one or more FcγR are used as fusionpartners with one or more specific NCSMs of the present invention andare useful in the applications described above.

In addition to fusion with Ig sequences or regions, the fusion proteinsof the invention can include any protein sequence in combination withthe NCSM molecule, or fragment thereof. The invention also includes thenucleic acid sequence encoding any such fusion polypeptide. For example,a NCSM polypeptide of the invention or fragments thereof can be fusedwith polypeptide sequences which, e.g., enable sorting of the fusionproteins, e.g., fluorescence indicator molecules. Alternatively, theNCSM polypeptides or fragments thereof can be incorporated into fusionproteins that enable, e.g., targeting of the fusions to specific celltypes or cells.

The fusion proteins of the present invention can be delivered to asubject by a variety of formats, including, e.g., as a polypeptide orprotein, or as a nucleic acid encoding such polypeptide or protein asdescribed in detail herein.

Therapeutic and Prophylactic Treatment Methods

The NCSM polynucleotides and polypeptides of the invention haveproperties that are of beneficial use in a variety of application,including, e.g., protein- and DNA-based vaccinations and in prophylacticand therapeutic disease treatments where manipulation of an immuneresponse (e.g., inducing or suppressing), T cell activation orproliferation, and/or cytokine production is desirable.

In one aspect, the present invention includes methods of therapeuticallyor prophylactically treating a disease or disorder by administering, invivo or ex vivo, one or more nucleic acids or fragments thereof orpolypeptides or fragments thereof of the invention described above (orcompositions, vectors, or transduced cells comprising a pharmaceuticallyacceptable excipient and one or more such nucleic acids or polypeptides)to a subject or to a population of cells of the subject, including,e.g., a mammal, including, e.g., a human, primate, monkey, orangutan,baboon, mouse, pig, cow, cat, goat, rabbit, rat, guinea pig, hamster,horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., achicken or duck) or a fish, or invertebrate.

In one aspect of the invention, in ex vivo methods, one or more cells ora population of cells of interest of the subject (e.g., tumor cells,tumor tissue sample, organ cells, blood cells, cells of the skin, lung,heart, muscle, brain, mucosae, liver, intestine, spleen, stomach,lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) areobtained or removed from the subject and contacted with an amount of apolypeptide of the invention that is effective in prophylactically ortherapeutically treating a disease, disorder, or other condition. Thecontacted cells are then returned or delivered to the subject to thesite from which they were obtained or to another site (e.g., includingthose defined above) of interest in the subject to be treated. Ifdesired, the contacted cells may be grafted onto a tissue, organ, orsystem site (including all described above) of interest in the subjectusing standard and well-known grafting techniques or, e.g., delivered tothe blood or lymph system using standard delivery or transfusiontechniques.

The CD28BP polypeptides of the invention and/or nucleic acids of theinvention can be used in methods to activate T cells ex vivo by, e.g.,obtaining or removing T cells from a subject (e.g., mammal, such as ahuman) and administering to the subject a sufficient amount of one ormore polypeptides of the invention to activate effectively the T cells(or administering a sufficient amount of one or more nucleic acids ofthe invention with a promoter such that uptake of the nucleic acid intoone or more such T cells occurs and sufficient expression of the nucleicacid results to produce an amount of a polypeptide effective to activatesaid T cells. The activated T cells are then returned to the subject. Tcells can be obtained or isolated from the subject by a variety ofmethods known in the art, including, e.g., by deriving T cells fromperipheral blood of the subject or obtaining T cells directly from atumor of the subject.

The CD28BP polypeptides of the invention and/or nucleic acids of theinvention can be used to activate T cells ex vivo by, e.g., obtaining orremoving cells (e.g., antigen presenting cells) from a subject (e.g., amammal, such as a human) and administering to the removed cells asufficient amount of one or more polypeptides of the invention toactivate effectively T cells once the removed cells are returned to thesubject (or administering a sufficient amount of one or more nucleicacids of the invention with a promoter such that uptake of the nucleicacid into one or more removed cells occurs and sufficient expression ofthe nucleic acid results to produce an amount of a polypeptide effectiveto activate T cells upon return of the removed cells to the subject).

The CTLA-4BP polypeptides of the invention and/or nucleic acids encodingpolypeptides of the invention are useful in inhibiting T cell response(e.g., inhibiting T cell activation or proliferation) in a subject towhich at least one at the polypeptides or nucleic acids of the inventionis administered. In another aspect, the CTLA-4BP polypeptides of theinvention and/or nucleic acids encoding polypeptides of the inventionmodulate T cell activation without completely inhibiting T cellproliferation following administration. In another aspect, the CTLA-4BPpolypeptides of the invention and/or nucleic acids encoding polypeptidesof the invention modulate T cell activation in a subject followingadministration, but do not induce proliferation of purified T cellsactivated by soluble monoclonal antibodies (e.g., anti-CD3 monoclonalantibodies that bind T cell receptor (TCR) on a T cell).

The invention also provides in vivo methods in which at least one cellor a population of cells of interest of the subject are contacteddirectly or indirectly with a sufficient amount of a NCSM polypeptide ofthe invention effective in prophylactically or therapeutically treatinga disease, disorder, or other condition. In direct (e.g., local) contactor administration formats, the polypeptide is typically administered ortransferred directly (e.g., locally) to the cells to be treated or tothe tissue site of interest (e.g., tumor cells, tumor tissue sample,organ cells, blood cells, cells of the skin, lung, heart, muscle, brain,mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix,vagina, prostate, mouth, tongue, etc.) by any of a variety of formats,including topical administration, injection (e.g., using a needle orsyringe), or vaccine or gene gun delivery, or pushing into a tissue,organ, or skin site.

The NCSM molecule can be delivered by a variety of routes, e.g.,intramuscularly, intradermally, subdermally, subcutaneously, orally,intraperitoneally, intrathecally, intravenously, mucosally,systemically, parenterally, via inhalation, or placed within a cavity ofthe body (including, e.g., during surgery), or by inhalation or vaginalor rectal administration.

In in vivo and ex vivo indirect contact/administration formats, the NCSMpolypeptide is typically administered or transferred indirectly to thecells to be treated or to the tissue site of interest, including thosedescribed above (such as, e.g., skin cells, organ systems, lymphaticsystem, or blood cell system, etc.), by contacting or administering theNCSM polypeptide of the invention directly to one or more cells orpopulation of cells from which treatment can be facilitated. Forexample, tumor cells within the body of the subject can be treated bycontacting cells of the blood or lymphatic system, skin, or an organwith a sufficient amount of the polypeptide such that delivery of thepolypeptide to the site of interest (e.g., tissue, organ, or cells ofinterest or blood or lymphatic system within the body) occurs andeffective prophylactic or therapeutic treatment results. Such contact,administration, or transfer is typically made by using one or more ofthe routes or modes of administration described above.

In another aspect, the invention provides ex vivo methods in which oneor more cells of interest or a population of cells of interest of thesubject (e.g., tumor cells, tumor tissue sample, organ cells, bloodcells, cells of the skin, lung, heart, muscle, brain, mucosae, liver,intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate,mouth, tongue, etc.) are obtained or removed from the subject andtransformed by contacting said one or more cells or population of cellswith a polynucleotide construct comprising a target nucleic acidsequence of the invention or fragments thereof, that encodes abiologically active polypeptide of interest (e.g., a polypeptide of theinvention) that is effective in prophylactically or therapeuticallytreating the disease, disorder, or other condition. The one or morecells or population of cells is contacted with a sufficient amount ofthe polynucleotide construct and a promoter controlling expression ofsaid nucleic acid sequence such that uptake of the polynucleotideconstruct (and promoter) into the cell(s) occurs and sufficientexpression of the target nucleic acid sequence of the invention resultsto produce an amount of the biologically active polypeptide effective toprophylactically or therapeutically treat the disease, disorder, orcondition. The polynucleotide construct may include a promoter sequence(e.g., WT, recombinant, or chimeric CMV promoter sequence) that controlsexpression of a NCSM nucleic acid sequence of the invention and/or, ifdesired, one or more additional nucleotide sequences encoding at leastone of another NCSM polypeptide, a cytokine, an adjuvant, or aco-stimulatory molecule, or other polypeptide of interest.

Following transfection, the transformed cells are returned, delivered,or transferred to the subject to the tissue site or system from whichthey were obtained or to another site (e.g., tumor cells, tumor tissuesample, organ cells, blood cells, cells of the skin, lung, heart,muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphaticsystem, cervix, vagina, prostate, mouth, tongue, etc.) to be treated inthe subject. If desired, the cells may be grafted onto a tissue, skin,organ, or body system of interest in the subject using standard andwell-known grafting techniques or delivered to the blood or lymphaticsystem using standard delivery or transfusion techniques. Such delivery,administration, or transfer of transformed cells is typically made byusing one or more of the routes or modes of administration describedabove. Expression of the target nucleic acid occurs naturally or can beinduced (as described in greater detail below) and an amount of theencoded polypeptide is expressed sufficient and effective to treat thedisease or condition at the site or tissue system.

In another aspect, the invention provides in vivo methods in which oneor more cells of interest or a population of cells of the subject (e.g.,including those cells and cell(s) systems and subjects described above)are transformed in the body of the subject by contacting the cell(s) orpopulation of cells with (or administering or transferring to thecell(s) or population of cells using one or more of the routes or modesof administration described above) a polynucleotide construct comprisinga nucleic acid sequence of the invention that encodes a biologicallyactive polypeptide of interest (e.g., a polypeptide of the invention)that is effective in prophylactically or therapeutically treating thedisease, disorder, or other condition.

The polynucleotide construct can be directly administered or transferredto cell(s) exhibiting or having the disease or disorder (e.g., by directcontact using one or more of the routes or modes of administrationdescribed above). Alternatively, the polynucleotide construct can beindirectly administered or transferred to cell(s) exhibiting or havingthe disease or disorder by first directly contacting non-diseasedcell(s) or other diseased cells using one or more of the routes or modesof administration described above with a sufficient amount of thepolynucleotide construct comprising the nucleic acid sequence encodingthe biologically active polypeptide, and a promoter controllingexpression of the nucleic acid sequence, such that uptake of thepolynucleotide construct (and promoter) into the cell(s) occurs andsufficient expression of the nucleic acid sequence of the inventionresults to produce an amount of the biologically active polypeptideeffective to prophylactically or therapeutically treat the disease ordisorder, and whereby the polynucleotide construct or the resultingexpressed polypeptide is transferred naturally or automatically from theinitial delivery site, system, tissue or organ of the subject's body tothe diseased site, tissue, organ or system of the subject's body (e.g.,via the blood or lymphatic system). Expression of the target nucleicacid occurs naturally or can be induced (as described in greater detailbelow) such that an amount of the encoded polypeptide expressed issufficient and effective to treat the disease or condition at the siteor tissue system. The polynucleotide construct may include a promotersequence (e.g., wild-type, recombinant or chimeric CMV promotersequence) that controls expression of the nucleic acid sequence and/or,if desired, one or more additional nucleotide sequences encoding atleast one of another NCSM polypeptide, a cytokine, an adjuvant, or aco-stimulatory molecule, or other polypeptide of interest.

In one aspect, tumor cells of a patient are transfected with a DNAplasmid vector encoding a NCSM polypeptide of interest (e.g., CD28BP) tofacilitate an improved immune response, (e.g., enhanced T cell responseor increased antibody titer). The tumor cells may be removed from thepatient and transfected ex vivo, and then re-delivered to the patient,preferably at the tumor site. Alternatively, the tumor cells of a tumorare transfected in vivo, by delivering a DNA plasmid encoding a NCSMpolypeptide of interest (e.g., CD28BP). In either case, the immuneresponse can be measured by measuring T cell proliferation using methodsdescribed herein or antibody levels using standard protocols. In anotheraspect, a DNA plasmid encoding a soluble NCSM-ECD or soluble NCSM-ECD-Igis administered to a patient by any means described herein, includingsystemically, subcutaneously, i.m., intradermally, etc. and the like,via a needle or gene gun or other introduction mechanism describedherein; if desired, the plasmid is introduced directly into cells of atumor or tumor-related cells of the patient.

In yet another aspect, a soluble NCSM-ECD polypeptide or solubleNCSM-ECD-Ig fusion protein is administered to a patient by any meansdescribed herein, including systemically, subcutaneously, i.m.,intradermally, etc. and the like, via a needle or gene gun or otherintroduction mechanism described herein; if desired, the polypeptide orfusion protein is introduced directly into cells of a tumor ortumor-related cells of the patient. The soluble NCSM can be administeredin conjunction with an antigen (either simultaneously or consecutively)as part of a vaccine protocol.

The NCSM polypeptides of the invention and the NCSM polynucleotidesencoding them are also useful as vaccine adjuvants in vaccineapplications as discussed herein and for diagnostic purposes, as for invitro applications for testing and diagnosing such diseases. Forexample, a polynucleotide encoding a NCSM of the invention, (e.g.,CD28BP) or an NCSM polypeptide (or fragment thereof, e.g., ECD, orfusion protein) can serve as an adjuvant to a DNA vaccine or proteinvaccine by enhancing immune-stimulating properties of the antigenencoded by the DNA vaccine or the protein antigen itself, respectively.In any of these formats, the NCSM molecule that results maynon-specifically enhance the immune response of the subject to anantigen.

In each of the in vivo and ex vivo treatment methods as described above,a composition comprising an excipient and the NCSM polypeptide ornucleic acid of the invention can be administered or delivered. In oneaspect, a composition comprising a pharmaceutically acceptable excipient(e.g., PBS) and a NCSM polypeptide or nucleic acid of the invention isadministered or delivered to the subject as described above in an amounteffective to treat the disease or disorder.

In another aspect, in each in vivo and ex vivo treatment methoddescribed above, the amount of polynucleotide administered to thecell(s) or subject can be an amount sufficient that uptake of saidpolynucleotide into one or more cells of the subject occurs andsufficient expression of said nucleic acid sequence results to producean amount of a biologically active NCSM polypeptide (e.g., ECD)effective to enhance an immune response in the subject, including animmune response induced by an immunogen (e.g., antigen). In anotheraspect, for each such method, the amount of polypeptide administered tocell(s) or subject can be an amount sufficient to enhance an immuneresponse in the subject, including that induced by an immunogen (e.g.,antigen).

In yet another aspect, in each in vivo and ex vivo treatment methoddescribed above, the amount of polynucleotide administered to thecell(s) or subject can be an amount sufficient that uptake of saidpolynucleotide into one or more cells of the subject occurs andsufficient expression of said nucleic acid sequence results to producean amount of a biologically active polypeptide effective to produce atolerance or anergy response in the subject. In another aspect, for eachsuch method, the amount of polypeptide administered to cell(s) orsubject can be an amount sufficient to produce a tolerance or anergyresponse in the subject.

The amount of DNA plasmid for use in such methods where administrationis by injection is from about 50 micrograms (ug) to 5 mg, usually about100 ug to about 2.5 mg, typically about 500 ug to 2 mg or about 800 ugto about 1.5 mg, and often about 1 mg. The amount of DNA plasmid for usein these methods where administration is via a gene gun, e.g., is fromabout 100 to 1000 times less; thus, for each range given above for DNAplasmid administration via injection, the range for DNA plasmidadministration via gene gun is about 100 to 1000 times less. Forexample, for gene gun delivery, the amount of DNA plasmid correspondingto the first range above is from about 50×10⁻⁸ g to 5×10⁻⁵ g (100 timesless) or from about 50×10⁻⁹ to about 5×10⁻⁶ g. DNA plasmid amounts canbe readily adjusted by those of ordinary skill in the art based uponresponses in animal models obtained using the DNA plasmid vectorencoding WT hB7-1 and/or antigen or based upon known DNA vaccinationstudies using plasmid vectors encoding a mammalian B7-1, such as WThB7-1. Such amounts of DNA plasmid can be used, if desired, in themethod in Example VI.

In yet another aspect, in an in vivo or in vivo treatment method inwhich a polynucleotide construct (or composition comprising apolynucleotide construct) is used to deliver a physiologically activepolypeptide to a subject, the expression of the polynucleotide constructcan be induced by using an inducible on- and off-gene expression system.Examples of such on- and off-gene expression systems include the Tet-On™Gene Expression System and Tet-Off™ Gene Expression System (see, e.g.,Clontech Catalog 2000, pg. 110–111 for a detailed description of eachsuch system), respectively. Other controllable or inducible on- andoff-gene expression systems are known to those of ordinary skill in theart. With such system, expression of the target nucleic of thepolynucleotide construct can be regulated in a precise, reversible, andquantitative manner. Gene expression of the target nucleic acid can beinduced, for example, after the stable transfected cells containing thepolynucleotide construct comprising the target nucleic acid aredelivered or transferred to or made to contact the tissue site, organ orsystem of interest. Such systems are of particular benefit in treatmentmethods and formats in which it is advantageous to delay or preciselycontrol expression of the target nucleic acid (e.g., to allow time forcompletion of surgery and/or healing following surgery; to allow timefor the polynucleotide construct comprising the target nucleic acid toreach the site, cells, system, or tissue to be treated; to allow timefor the graft containing cells transformed with the construct to becomeincorporated into the tissue or organ onto or into which it has beenspliced or attached, etc.).

The present invention also provides a therapeutic method of activatingor enhancing a T cell response in a subject suffering from a cancer,such as, e.g., where the subject has a tumor. The method comprisesadministering to the subject a composition that comprises a nucleotidesequence that encodes a soluble NCSM polypeptide and an excipient,wherein the NCSM polypeptide is expressed by the tumor cells or thetumor-related cells, and the T cell response is activated or enhancedagainst the tumor. The composition may be a pharmaceutical composition,and the excipient may be a pharmaceutically acceptable excipient. Thepharmaceutical composition may comprise a nucleotide sequence encoding asoluble NCSM polypeptide (or fragment thereof having at least one NCSMproperty) and a pharmaceutically acceptable excipient. Such nucleotidesequence may be incorporated in a vector and may be operably linked to apromoter to facilitate expression.

In one embodiment, the composition comprising a nucleotide sequenceencoding a soluble NCSM polypeptide (or fragment thereof having at leastone NCSM property) and an excipient is administered to the subject byi.d., i.m. or, e.g., direct injection or via gene gun or other vaccinedelivery device. The composition may be introduced or administered by avariety of routes, including, direct administration to the tumor ortumor site, if known, or administration systemically to the subject bydirect injection or gene gun or the like. A sufficient amount of thecomposition is delivered such that transfection of the subject's tumorcells with the NCSM-polypeptide-encoding nucleotide sequence occurs anda T cell response or activation results. As described above, a DNAplasmid expression vector comprising the nucleotide sequence may bedelivered as “naked” DNA or may be formulated with other components(e.g., calcium phosphate, lipids, etc.) to facilitate transfection.Exemplary amounts of the total DNA (e.g., in milligrams) (for the NCSMpolynucleotide and vector) suggested for such treatment are describedherein and in the Examples below. The amount of DNA plasmid may be atherapeutically effective amount to inhibit further growth of the tumoror kill the tumor. One of skill in the art can also determine atherapeutically effective DNA plasmid vector amounts based on knownclinical studies to treat cancers using gene therapy or DNA vaccinationmethods and WT hB7-1 and mammalian models.

In another aspect, tumor cells are obtained from the subject (oralternatively allogeneic tumor cells are used). The tumor cells aretransfected using techniques described herein) with a sufficient amountof an expression vector, such as e.g., a pMax Vax vector, described inthe Example V below, that comprises a NCSM polynucleotide encoding aNCSM polypeptide (full-length) or soluble NCSM-ECD polypeptide (orNCSM-ECD-Ig fusion protein) such that expression results, and in thecase of soluble polypeptides, the soluble polypeptides are secreted.

Genetic Vectors

Gene therapy and genetic vaccine vectors are useful for treating and/orpreventing various diseases and other conditions. The followingdiscussion focuses on the on the use of vectors because gene therapy andgenetic vaccine method typically employ vectors, but persons of skill inthe art appreciate that the nucleic acids of the invention can,depending on the particular application, be employed in the absence ofvector sequences. Accordingly, references in the following discussion tovectors should be understood as also relating to nucleic acids of theinvention that lack vector sequences. The invention includes vectorscomprising one or more NCSM nucleic acids of the invention, includingnucleic acids encoding B7-1 and B7-2 polypeptide variants as describedherein, including, e.g., variants of human, primate, and bovine B7-1 andB7-2.

Vectors can be delivered to a subject to induce an immune response orother therapeutic or prophylactic response. Suitable subjects include,but are not limited to, a mammal, including, e.g., a human, primate,monkey, orangutan, baboon, mouse, pig, cow, cat, goat, rabbit, rat,guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such asa bird (e.g., a chicken or duck) or a fish, or invertebrate.

Vectors can be delivered in vivo by administration to an individualpatient, typically by local (direct) administration or by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,subdermal, intracranial, anal, vaginal, oral, mucosal, inhalation,systemic, parenteral, buccal route or they can be inhaled) or they canbe administered by topical application. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

In local (direct) administration formats, the nucleic acid or vector istypically administered or transferred directly to the cells to betreated or to the tissue site of interest (e.g., tumor cells, tumortissue sample, organ cells, blood cells, cells of the skin, lung, heart,muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphaticsystem, cervix, vagina, prostate, mouth, tongue, etc.) by any of avariety of formats, including topical administration, injection (e.g.,by using a needle or syringe), or vaccine or gene gun delivery, pushinginto a tissue, organ, or skin site. For standard gene gunadministration, the vector or nucleic acid of interest is precipitatedonto the surface of microscopic metal beads. The microprojectiles areaccelerated with a shock wave or expanding helium gas, and penetratetissues to a depth of several cell layers. For example, the Accel™ GeneDelivery Device manufactured by Agacetus, Inc. Middleton Wis. issuitable for use in this embodiment. The nucleic acid or vector can bedelivered, for example, intramuscularly, intradermally, subdermally,subcutaneously, orally, intraperitoneally, intrathecally, intravenously,mucosally, systemically, parenterally, via inhalation, or placed withina cavity of the body (including, e.g., during surgery), or by inhalationor vaginal or rectal administration.

In in vivo indirect contact/administration formats, the nucleic acid orvector is typically administered or transferred indirectly to the cellsto be treated or to the tissue site of interest, including thosedescribed above (such as, e.g., skin cells, organ systems, lymphaticsystem, or blood cell system, etc.), by contacting or administering thenucleic acid or vector of the invention directly to one or more cells orpopulation of cells from which treatment can be facilitated. Forexample, tumor cells within the body of the subject can be treated bycontacting cells of the blood or lymphatic system, skin, or an organwith a sufficient amount of the polypeptide such that delivery of thenucleic acid or vector to the site of interest (e.g., tissue, organ, orcells of interest or blood or lymphatic system within the body) occursand effective prophylactic or therapeutic treatment results. Suchcontact, administration, or transfer is typically made by using one ormore of the routes or modes of administration described above.

A large number of delivery methods are well known to those of skill inthe art. Such methods include, for example liposome-based gene delivery(Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988)BioTechniques 6(7):682–691; Rose U.S. Pat No. 5,279,833; Brigham (1991)WO 91/06309; and Felgner et al. (1987) Proc. Natl Acad. Sci. USA84:7413–7414), as well as use of viral vectors (e.g., adenoviral (see,e.g., Berns et al. (1995) Ann. NY Acad. Sci. 772:95–104; Ali et al.(1994) Gene Ther. 1:367–384; and Haddada et al. (1995) Curr. Top.Microbiol. Immunol. 199 (Pt 3):297–306 for review), papillomaviral,retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731–2739; Johann et al. (1992) J. Virol. 66 (5):1635–1640 (1992);Sommerfelt et al., (1990) Virol. 176:58–59; Wilson et al. (1989) J.Virol. 63:2374–2378; Miller et al., J. Virol. 65:2220–2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al., Gene Therapy (1994)supra.), and adeno-associated viral vectors (see, West et al. (1987)Virology 160:38–47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carteret al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793–801;Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for anoverview of AAV vectors; see also, Lebkowski, U.S. Pat. No. 5,173,414;Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251–3260; Tratschin, etal. (1984) Mol. Cell. Biol., 4:2072–2081; Hermonat and Muzyczka (1984)Proc. Natl Acad. Sci. USA, 81:6466–6470; McLaughlin et al. (1988) andSamulski et al. (1989) J. Virol., 63:03822–3828), and the like.

“Naked” DNA and/or RNA that comprises a genetic vaccine can also beintroduced directly into a tissue, such as muscle, by injection using aneedle or other similar device. See, e.g., U.S. Pat. No. 5,580,859.Other methods such as “biolistic” or particle-mediated transformation(see, e.g., Sanford et al., U.S. Pat. Nos. 4,945,050; 5,036,006) arealso suitable for introduction of genetic vaccines into cells of amammal according to the invention. These methods are useful not only forin vivo introduction of DNA into a subject, such as a mammal, but alsofor ex vivo modification of cells for reintroduction into a mammal. DNAis conveniently introduced directly into the cells of a mammal or othersubject using, e.g., injection, such as via a needle, or a “gene gun.”As for other methods of delivering genetic vaccines, if necessary,vaccine administration is repeated in order to maintain the desiredlevel of immunomodulation, such as the level or response of T cellactivation or T cell proliferation, or antibody titer level.Alternatively, nucleotides can be impressed into the skin of thesubject.

Gene therapy and genetic vaccine vectors (e.g., DNA, plasmids,expression vectors, adenoviruses, liposomes, papillomaviruses,retroviruses, etc.) comprising at least one NCSM sequence can beadministered directly to the subject (usually a mammal) for transductionof cells in vivo or ex vivo. The vectors can be formulated aspharmaceutical compositions for administration in any suitable manner,including parenteral (e.g., subcutaneous, intramuscular, intradermal, orintravenous), inhalation, mucosal, topical, oral, rectal, vaginal,intrathecal, buccal (e.g., sublingual), or local administration, such asby aerosol or transdermally, for immunotherapeutic or other prophylacticand/or therapeutic treatment. Pretreatment of skin, for example, by useof hair-removing agents, may be useful in transdermal delivery. Suitablemethods of administering such packaged nucleic acids are available andwell known to those of skill in the art, and, although more than oneroute can be used to administer a particular composition, a particularroute can often provide a more immediate and more effective reactionthan another route.

Further, the vectors of this invention comprising at least onenucleotide sequence encoding at least one NCSM (and, if desired, furthercomprising a nucleotide sequence encoding antigen or otherco-stimulatory molecule co-expressed on the same vector) can be used toprophylactically or therapeutically treat or supplement such treatmentof other immunological disorders and diseases or enhance protectionagainst disorders, diseases, and antigens (including WT and recombinantantigens), e.g., in protein vaccines and DNA vaccines, including, butnot limited to, e.g., allergy/asthma, neurological, organtransplantation (e.g., graft versus host disease, and autoimmunediseases), malignant diseases, chronic infectious diseases, including,but not limited to, e.g., viral infectious diseases, such as thoseassociated with, but not limited to, e.g., alpha viruses, hepatitisviruses, e.g., hepatitis B virus (HBV), herpes simplex virus (HSV),hepatitis C virus (HCV), HIV, human papilloma virus (HPV), malaria,Venezuelan equine encephalitis (VEE), Western equine encephalitis (WEE),Japanese encephalitis virus, Eastern equine encephalitis, and the like,and bacterial infectious diseases, such as, e.g., but not limited to,e.g., Lyme disease, tuberculosis, and chlamydia infections; and otherdiseases and disorders described herein.

If desired, a separate vector comprising a nucleotide sequence encodingan antigen or other co-stimulatory molecule can be deliveredsimultaneously with a vector comprising a NCSM sequence of theinvention.

Compositions and Formulations

The present invention also includes compositions of any NSCM nucleicacid or NCSM polypeptide of the invention, including B7-1 and B7-2variant nucleic acids and polypeptide variants, including, e.g., nucleicacid variants and polypeptide variants of human, primate, and bovineB7-1 and B7-2. The invention also includes compositions comprising oneor more vectors or cells (or a population of cells) comprising any suchpolypeptide or nucleic acid of the invention. In one aspect, theinvention provides therapeutic and/or prophylactic compositionscomprising at least one NCSM polypeptide (or fragment thereof) ornucleic acid (or fragment thereof) of the invention, or vectors,transduced cells, or vaccines comprising at least one NCSM nucleic acidor polypeptide (or fragment) of the invention. Such compositionsoptionally are tested in appropriate in vitro and in vivo animal modelsof disease, to confirm efficacy, tissue metabolism, and to estimatedosages, according to methods well known in the art. In particular,dosages for therapeutic and prophylactic methods for treating orpreventing a disease or condition can be determined by activitycomparison of the NCSM molecules to other known therapeutics usingsimilar compositions in a relevant assay and mammalian model, includingas described below.

Administration optionally is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cells.See, supra. The NCSM polypeptides and polynucleotides, and vectors,cells, and compositions comprising such molecules, are administered inany suitable manner, preferably with pharmaceutically acceptablecarriers. Suitable methods of administering such NCSM molecules, in thecontext of the present invention, to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route. Preferred routes are readilyascertained by those of skill in the art.

Compositions comprising cells expressing at least one full length formof a NCSM polypeptide or a fragment thereof (ECD) are also a feature ofthe invention. Such cells may also express one or more antigens specificfor the intended application (e.g., cancer antigen). Such cells arereadily prepared as described herein by transfection with DNA plasmidvector encoding at least one of the NCSM polypeptide and/or antigen.Separate vectors each encoding a NCSM polypeptide and antigen may beused to transfect the cells, or a bicistronic vector encoding both theNCSM polypeptide and antigen can be used. Compositions of such cells maybe pharmaceutically compositions further comprising a pharmaceuticallyacceptable carrier or excipient.

Pharmaceutical compositions of the invention can, but need not, includea pharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention. Avariety of aqueous carriers can be used, e.g., buffered saline, such asPBS, and the like. These solutions are sterile and generally free ofundesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of gene therapy or geneticvaccine vector in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Compositions comprising NCSM polypeptides and polynucleotides, andvectors, cells, and other formulations comprising these and othercomponents of the invention, can be administered by a number of routesincluding, but not limited to oral, intranasal, intravenous,intraperitoneal, intramuscular, transdermal, subcutaneous, intradermal,topical, systemic, mucosal, inhalation, parenteral, sublingual, vaginal,or rectal means. Polypeptide and nucleic acid compositions can also beadministered via liposomes. Such administration routes and appropriateformulations are generally known to those of skill in the art.

The NCSM polypeptide or polynucleotide or fragment thereof, or vectorcomprising a NCSM nucleic acid, alone or in combination with othersuitable components, can also be made into aerosol formulations (e.g.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art. It is recognized that the gene therapy vectors andgenetic vaccines, when administered orally, must be protected fromdigestion. This is typically accomplished either by complexing thevector with a composition to render it resistant to acidic and enzymatichydrolysis or by packaging the vector in an appropriately resistantcarrier such as a liposome. Means of protecting vectors from digestionare well known in the art. The pharmaceutical compositions can beencapsulated, e.g., in liposomes, or in a formulation that provides forslow release of the active ingredient.

The packaged nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (e.g., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, subdermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by intravenous infusion, orally,mucosally, topically, intraperitoneally, intravesically orintrathecally. The formulations of packaged nucleic acids orpolypeptides of the invention can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, any routes of administrationalready in use for existing co-stimulatory therapeutics and prophylactictreatment protocols, including those currently employed with e.g.,mammalian B7-1 polynucleotides and polypeptides, such as hB7-1, alongwith pharmaceutical compositions and formulations in current use, arepreferred routes of administration and formulation for the NCSMpolynucleotides or polypeptides (and fragments thereof).

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid can also be administeredintravenously or parenterally.

Cells transduced with the NCSM nucleic acids as described herein in thecontext of ex vivo or in vivo therapy can also be administeredintravenously or parenterally. It will be appreciated that the deliveryof cells to patients is routine, e.g., delivery of cells to the bloodvia intravenous, intramuscular, or intraperitoneal administration orother common route.

The dose administered to a patient, in the context of the presentinvention is sufficient to effect a beneficial effect, such as analtered immune response or other therapeutic and/or prophylacticresponse in the patient over time, or to, e.g., inhibit infection by apathogen, depending on the application. The dose will be determined bythe efficacy of the particular nucleic acid, polypeptide, vector,composition or formulation, transduced cell, cell type, and/or theactivity of the NCSM polypeptide and/or polynucleotide included thereinor employed, and the condition of the patient, as well as the bodyweight, surface area, or vascular surface area, of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of any such particular polypeptide, nucleic acid, vector,formulation, composition, transduced cell, cell type, or the like in aparticular patient. Dosages to be used for therapeutic or prophylactictreatment of a particular disease or disorder can be determined by oneof skill by comparison to those dosages used for existing therapeutic orprophylactic treatment protocols for the same disease or disorder.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid can also be administeredintravenously or parenterally.

In determining the effective amount of the vector, cell type,composition, or formulation to be administered to a subject for thetreatment or prophylaxis of the medical condition or disease state(e.g., cancers or viral diseases), a physician evaluates the subjectfor, e.g., circulating plasma levels, vector/cell/formulation/NCSMmolecule toxicities, progression of the disease or condition, and theproduction of anti-vector/NCSM polypeptide antibodies, and depending onthe subject other factors that would be known to one of skill in theart.

In one aspect, for example, in determining the effective amount of thevector to be administered in the treatment or prophylaxis of aninfection or other condition, wherein the vector comprises any NCSMnucleic acid sequence described herein or encodes any NCSM polypeptidedescribed herein, the physician evaluates vector toxicities, progressionof the disease, and the production of anti-vector antibodies, if any. Inone aspect, the dose equivalent of a naked nucleic acid from a vectorfor a typical 70 kilogram patient can range from about 10 ng to about 1g, about 100 ng to about 100 mg, about 1 μg to about 10 mg, about 10 μgto about 1 mg, or from about 30–300 μg. Doses of vectors used to deliverthe nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses.

In another aspect, the dose administered, e.g., to a 70 kilogram patientcan be in the range equivalent to any dosages of currently-usedco-stimulatory or WT B7-1 therapeutic or prophylactic proteins (such ahB7-1) or the like, and doses of vectors or cells which produce NCSMsequences optionally are calculated to yield an equivalent amount ofNCSM nucleic acid or expressed polypeptide or protein. The vectors ofthis invention comprising at least one nucleotide sequence encoding atleast one NCSM (and, if desired, further comprising a nucleotidesequence encoding antigen or other co-stimulatory molecule either on thesame vector) can be used to prophylactically or therapeutically treat orsupplement such treatment of a variety of cancers, including e.g.,colorectal cancer, breast cancer, pancreatic cancer, lung cancer,prostate cancer, naso-pharyngeal cancer, cancer, brain cancer, leukemia,melanoma, head- and neck cancer, stomach cancer, cervical cancer,ovarian cancer, lymphomas, colon cancer, colorectal, andvirally-mediated conditions by any known conventional therapy, includingcytotoxic agents, nucleotide analogues (e.g., when used for treatment ofHIV infection), biologic response modifiers, and the like.

In therapeutic applications, compositions are administered to a patientsuffering from a disease (e.g., an infectious disease, cancer, orautoimmune disorder) in an amount sufficient to cure or at leastpartially arrest or ameliorate the disease or at least one of itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of protein to effectively treat thepatient.

In prophylactic applications, compositions are administered to a humanor other mammal to induce an immune or other prophylactic response thatcan help protect against the establishment of an infectious disease,cancer, autoimmune disorder, or other condition.

In some applications, an amount of NCSM polypeptide that is administeredto a subject for a particular therapeutic or prophylactic treatmentprotocol or vaccination ranges from about 1 to about 50 mg/kg weight ofthe subject. Such amount of polypeptide can be administered 1 time/weekor up to 3 times/week, as desired. Such NCSM polypeptide can beadministered as a soluble molecule comprising, e.g., an NCSM-ECD, orNCSM-trunECD-Ig or NCSM-ECD-Ig fusion protein. Alternatively, such NCSMpolypeptide can be administered in the form of aNCSM-polypeptide-encoding polynucleotide, which is operably linked to apromoter, such that the polynucleotide expresses in the subject such aNCSM polypeptide of from about 1 to about 50 mg/kg weight of the subject(e.g., on the surface of targeted cells) or as an expressed soluble NCSMpolypeptide. The NCSM polypeptide (or nucleic acid encoding thepolypeptide) can be administered to a population of cells of a subjectin vivo, or to a population of cells of the subject ex vivo as describedherein. Compositions comprising soluble NCSM polypeptides in such rangeamounts or comprising nucleic acids or expression vectors that canexpress such amounts in the subject are also contemplated.

In cancer immunotherapy or prophylactic applications (e.g., multiplemyeloma, breast cancer, lymphoma, and the like), it is advantageous toadminister at least one NCSM molecules (including, e.g., CD28BPs andCTLA-4BPs) and at least one cancer antigen with at least one othermolecule of interest, such as, e.g., a cytokine (IL-12, IL-15, IL-2, orvariant thereof, etc.) and/or colony stimulating factor (e.g., GM-CSF);such combination can serve to enhance a desired response, e.g., toenhance lymphocyte proliferation and/or gamma-interferon release.Included are recombinant, variant and mutant forms of IL-12, includingrecombinant IL-12p-40 and IL-12p35 polypeptides and nucleic acidsdescribed in PCT App. No. US00/32664 (Publ. No. WO 01/40257), which isincorporated herein by reference in its entirety for all purposes. Inone format, a bicistronic vector comprising nucleotide sequencesencoding an NCSM polypeptide, cancer antigen, and polypeptide(s) ofinterest is administered to the subject (e.g., by intramuscular orintradermal injection). In another format, a vector comprising anucleotide sequence encoding the molecule of interest can beadministered separately to the patient, at the same time or followingadministration of the one or more vectors comprising sequences encodingthe antigen and NCSM polypeptide. Typically, a dose of at least about 1mg nucleic acid (e.g., DNA) of GM-CSF and/or IL-2, IL-12 or othercytokine is administered at the time of immunization with the antigenand NCSM nucleic acids. Alternatively, the additional molecule ofinterest (GM-CSF, IL-12, IL-2, or other cytokine) is administered to thesubject as a polypeptide (e.g., by i.m. or i.d. injection). The initialdose of this polypeptide is administered at about the same time as thevector encoding the NCSM polypeptide and antigen, and typicallycomprises at least about 75 ug. Subsequent additional “boost” doses ofat least about 75 ug are usually delivered once/day for at least fourdays following the initial immunization. In another format, one or morevectors encoding either or both an NCSM polypeptide and molecule ofinterest (cytokine, GM-CSF) are administered (via, e.g., i.d. or i.m.injection) in vivo into the tumor of a subject where the tumor isinoperable, or into tumor cells removed from a patient (ex vivoadministration). Additional vector formats can also be used (adenoviral,retroviral, bicistronic, tricistronic).The toxicity and therapeuticefficacy of the vectors that include recombinant molecules provided bythe invention are determined using standard pharmaceutical procedures incell cultures or experimental animals. One can determine the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population) using procedurespresented herein and those otherwise known to those of skill in the art.Nucleic acids, polypeptides, proteins, fusion proteins, transduced cellsand other formulations of the present invention can be administered at arate determined, e.g., by the LD₅₀ of the formulation, and theside-effects thereof at various concentrations, as applied to the massand overall health of the patient. Again, administration can beaccomplished via single or divided doses.

A typical pharmaceutical composition for intravenous administration isabout 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100mg per patient per day may be used, particularly when the drug isadministered to a secluded site and not into the blood stream, such asinto a body cavity or into a lumen of an organ. Substantially higherdosages are possible in topical administration. For recombinantpromoters of the invention that express the linked transgene at highlevels, it may be possible to achieve the desired effect using lowerdoses, e.g., on the order of about 1 μg or 10 μg per patient per day.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

For introduction of recombinant NCSM nucleic acid transduced cells intoa patient, an illustrative, but not limiting example includes takingblood samples, obtained prior to infusion, and saved for analysis.Between, e.g., 1×10⁶ and 1×10¹² transduced cells are infusedintravenously over, e.g., 60–200 minutes. Vital signs and oxygensaturation by pulse oximetry are closely monitored. Blood samples areobtained, e.g., 5 minutes and, e.g., 1 hour following infusion and savedfor subsequent analysis. Leukopheresis, transduction and reinfusion areoptionally repeated every, e.g., 2 to 3 months for a total of, e.g., 4to 6 treatments in a one year period. After the first treatment,infusions can be performed, e.g., on a outpatient basis at thediscretion of the clinician. If the reinfusion is given as anoutpatient, the participant is monitored for, e.g., at least 4, andpreferably, e.g., 8 hours following the therapy. Transduced cells areprepared for reinfusion according to established methods. See,Abrahamsen et al. (1991) J Clin Apheresis 6:48–53; Carter et al. (1988)J Clin Arpheresis 4:113–117; Aebersold et al. (1988), J Immunol Methods112:1–7; Muul et al. (1987) J Immunol Methods 101:171–181 and Carter etal. (1987) Transfusion 27:362–365. After a period of, e.g., about 2–4weeks in culture, the cells should number between, e.g., 1×10⁶ and1×10¹². In this regard, the growth characteristics of cells vary frompatient to patient and from cell type to cell type. About, e.g., 72hours prior to reinfusion of the transduced cells, an aliquot is takenfor analysis of phenotype, and percentage of cells expressing thetherapeutic agent.

If a patient undergoing infusion of a vector or transduced cell orprotein formulation develops, e.g., fevers, chills, or muscle aches,he/she receives the appropriate dose of, e.g., aspirin, ibuprofen,acetaminophen or other pain/fever controlling drug. Patients whoexperience reactions to the infusion such as fever, muscle aches, andchills are premedicated, e.g., 30 minutes prior to the future infusionswith, e.g., either aspirin, acetaminophen, or, e.g., diphenhydramine,etc. Meperidine is used for more severe chills and muscle aches that donot quickly respond to antipyretics and antihistamines. Cell infusionis, e.g., slowed or discontinued depending upon the severity of thereaction.

The NCSM polypeptides, NCSM nucleic acids, and cells, vectors,transgenic animals, and compositions that include the NCSM molecules ofthe invention can be packaged in packs, dispenser devices, and kits foradministration to a subject, such as a mammal. For example, packs ordispenser devices that contain one or more unit dosage forms areprovided. Typically, instructions for administration of the compoundswill be provided with the packaging, along with a suitable indication onthe label that the compound is suitable for treatment of an indicatedcondition. For example, the label may state that the active compoundwithin the packaging is useful for treating a particular infectiousdisease, autoimmune disorder, tumor, or for preventing or treating otherdiseases or conditions that are mediated by, or potentially susceptibleto, a subject's or mammalian immune response.

Any NCSM nucleic acid, polypeptide, protein, fusion protein, or vectoror comprising any such NCSM molecule described herein, and anycomposition comprising at least one NCSM nucleic acid, polypeptide,protein, fusion protein, or vector or cell comprising at least one suchNCSM molecule, can be used in any of the methods and applicationsdescribed herein. In one aspect, the invention provides for the use ofany NCSM polypeptide or nucleic acid (or vector or cell comprising aNCSM nucleic acid) or composition thereof as a medicament, or as avaccine, for the treatment of one of the diseases described herein orfor preventing one of the diseases described herein, or the like. Inanother aspect, the invention provides for the use of any NCSMpolypeptide or nucleic acid (or vector or cell comprising a NCSM nucleicacid) or composition thereof for the manufacture of a medicament,prophylactic, therapeutic, drug, or vaccine, including for anytherapeutic or prophylactic application relating to treatment of adisease or disorder as described herein.

In one aspect, the invention provides methods for modulating or alteringan immune response T-cell response specific to an antigen in a subject.Some such methods comprise administering to the subject at least onepolynucleotide sequence comprising a NCSM polynucleotide described here(e.g., SEQ ID NOS:1–47, 95–173, and 253–262, 274–277) or at least onepolynucleotide encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272, 279–293 or fragment thereof, and apolynucleotide sequence encoding the antigen or antigenic fragmentthereof. Each of the at least one polynucleotide sequences is expressedin the subject in an amount effective to modulate or alter an immuneresponse or a T cell response. In some such methods, the polypeptide orfragment thereof interacts with or binds a T cell surface receptor. Insome such methods, T-cell response is enhanced as measured by assaysdescribed herein, and in some such methods, the enhanced T cell responseis sufficient to eliminate cells bearing the antigen or antigenicfragment thereof. In other methods, the T-cell response is suppressed orinhibited as measured by assays described herein.

In some such methods, the antigen or antigenic fragment thereof is anantigen or antigenic fragment thereof of an infectious agent or acancer. The encoded polypeptide may comprising any NCSM polypeptide offragment thereof described herein, such as SEQ ID NO:66 or SEQ ID NO:86,or the extracellular domain amino acid sequence of any NCSM polypeptidedescribed herein, or fusion protein thereof.

The at least one polynucleotide sequence encoding a NCSM polypeptide orfragment thereof may be operably linked to a promoter in a vector, suchas an expression vector or DNA plasmid. In one aspect, the at least onepolynucleotide sequence encoding the antigen or antigenic fragmentthereof may be included in the same vector and operably linked to asecond promoter in the same vector (e.g., bicistronic vector).Alternatively, the polynucleotide sequences encoding the NCSMpolypeptide and the antigen or antigenic fragment are present inseparate vectors and administered separately, e.g., eithersimultaneously or consecutively. The antigen or antigenic fragmentthereof may thus be operably linked to a promoter in the second vector.

In another aspect, the invention provides vectors comprising at leastone NCSM polynucleotide sequence described herein (e.g., SEQ ID NOS1–47,95–173, and 253–262) or a polynucleotide sequence encoding a polypeptidecomprising any of SEQ ID NOS:48–94, 174–252, 263–272 and 283–293 orfragment thereof, and a polynucleotide sequence encoding the antigen orantigenic fragment thereof, wherein the NCSM polypeptide or fragmentthereof interacts with or binds to a T cell receptor when expressed in asubject, and wherein each of the at least one polynucleotide sequencesis operably linked to a promoter for expression in the subject and ispresent in an amount sufficient that when expressed is effective tomodulate or alter a T cell response. In some such methods, the at leastone polynucleotide sequence encoding a polypeptide comprises apolynucleotide sequence of any of SEQ ID NOS:1–47, 95–173, and 253–262.Each of the at least one polynucleotide sequences may be expressed inthe subject in an amount effective to enhance a T cell response suchthat cells expressing the antigen or antigenic fragment thereof areeliminated. In some methods, each of the at least one polynucleotidesequences is expressed in the subject in an amount effective to inhibita T cell response.

In another aspect, the invention provides vectors comprising at leastone NCSM polynucleotide sequence described herein or at least onepolynucleotide sequence encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272 and 283–293 or fragment thereof, wherein thepolypeptide or fragment thereof interacts with or binds to a T cellreceptor when expressed in a subject, wherein the at least onepolynucleotide sequence is operably linked to a promoter for expressionin the subject and is present in an amount sufficient that whenexpressed is effective to modulate or alter a T cell response.

In yet another aspect, the invention provides methods of modulating oraltering an immune response in a subject, the method comprisingintroducing into cells of a tumor of the subject at least onepolynucleotide sequence encoding a polypeptide comprising any of SEQ IDNOS:48–94, 174–252, 263–272 and 283–293 or fragment thereof, wherein thepolypeptide or fragment thereof interacts with or binds to a T cellreceptor when expressed in a subject, and wherein the at least onepolynucleotide sequence is operably linked to a promoter for expressionin the subject and present in an amount sufficient that when expressedis effective to modulate or alter a T cell response.

The invention includes therapeutic methods for activating or enhancing aT-cell response in a subject, wherein the subject may have a tumor orfrom whom a tumor was surgically removed. Such methods compriseadministering to the subject a composition that comprises apolynucleotide sequence encodes a NCSM polypeptide and an excipient,wherein the NCSM polypeptide is expressed by tumor cells ortumor-related cells of the subject, and the T-cell response is activatedor enhanced against the tumor. For some such methods, the polynucleotidesequence encodes a soluble NCSM polypeptide. The composition maycomprise a vector comprising the polynucleotide sequence that encodes aNCSM polypeptide. Further, a therapeutically effective amount of thecomposition sufficient to enhance a T-cell response against the tumormay be administered. A pharmaceutical composition comprising anexpression vector comprising a polynucleotide sequence that encodes aNCSM polypeptide and a pharmaceutically acceptable excipient are alsoprovided.

The invention also includes therapeutic methods for activating orenhancing a T-cell response in a subject who has a tumor or from whom atumor was removed surgically, the method comprising administering to thesubject a composition that comprises a soluble NCSM polypeptide and anexcipient, wherein the T-cell response is activated or enhanced againstthe tumor. Also included are methods for activating or enhancing aT-cell response in such a subject, the methods comprising administeringto the subject a sufficient amount of a composition comprising anexcipient and a population of cells expressing a NCSM polypeptide and anantigen, such that the T-cell response is thereby activated or enhancedagainst the tumor. Also contemplated are methods for activating orenhancing a T-cell response in such a subject, the method comprisingadministering to the subject a sufficient amount of a compositioncomprising an excipient and a population of cells expressing a NCSMpolypeptide, such that the T-cell response is thereby activated orenhanced against the tumor.

Uses and Applications

The evolved novel NCSM molecules of the invention, in all formatsdescribed herein, including, but not limited to, e.g., NCSM nucleicacids, NCSM polypeptides and proteins, and vectors, cells, compositionsincluding such NCSM molecules, are useful in a broad range of clinical,therapeutic, and prophylactic applications. Optionally, the polypeptidesalone or fragments thereof are used to enhance the immune system (e.g.,NCSM polypeptides or soluble NCSM polypeptides (e.g., ECD), NCSM fusionproteins (e.g., comprising an NCSM-ECD fused to an Ig)). For example,these molecules are useful in enhancing tumor immunity and aspolypeptide or protein adjuvants and DNA vaccine adjuvants incombination with, e.g., antigens for specific diseases. For example,CD28BP polypeptides and nucleic acids encoding CD28BP polypeptides areuseful are adjuvants to enhance or boost an immune response in asubject, including to augment or enhance a response to a particularcompound that is delivered to the subject simultaneously or before orafter the CD28BP adjuvant. They are also useful in the treatment of avariety of medical conditions, including, e.g., chronic infectiousdiseases, allergies, autoimmune diseases, and in organ transplantationand the reversal of septic shock. Moreover, transgenic animals, such aspigs, mice, etc., expressing CD28BP and/or CTLA-4BP can be generatedusing methods known to those skilled in the art. Proteins, tissues ororgans from such animals can be used to modulate T cell responses inpatients undergoing tissue or organ transplantation.

Furthermore, NCSM molecules, such as the CD28BP and CTLA-4BP moleculesdescribed herein, are useful as components in multi-component vaccines,which optionally comprise, e.g., a single vector with multiplecomponents or multiple vectors encoding different vector components or amulti-component protein-based vaccines in which a CD28BP or CTLA-4BPprotein is delivered with other proteins, such as a protein vaccine. TheCD28BP or CTLA-4BP protein can be delivered simultaneously with theother protein(s) if desired, or delivered at a different time, and canalso be administered to a subject following delivery of a proteinvaccine or DNA vaccine to boost the immune response to the proteinvaccine or DNA vaccine. A multi-component vaccine optionally comprises,e.g., a vector, such as a DNA plasmid vector, that comprises, forexample, in addition to nucleotide sequences encoding one or more CD28BPand/or CTLA-4BP polypeptides, one or more nucleotide sequences encodingat least of the following components: at least one antigen(s),cytokine(s), adjuvant(s), promoter (e.g., wild-type CMV promoter (suchas human CMV promoter with or without an intron A sequence; or arecombinant, or chimeric CMV promoter with or without a recombinant orWT intron A sequence), and/or other co-stimulatory molecule(s) (each ofwhich may have been optimized by recursive sequence recombination andselection/screening procedures, random mutagenesis, or other knownmutagenesis procedures), and combinations of such various components.Such multi-component vector expresses two or more such components andincludes appropriate expression elements for such expression (see, e.g.,an exemplary multi-component vector described in Example V). Such anarrangement permits co-delivery of various components, includingrecursively recombined components, for a particular treatment regimen ortherapeutic or prophylactic application. Such vectors are designedaccording to the specific treatment regimen or therapeutic orprophylactic application desired. One or more such single-component ormulti-component vectors as described above may be used simultaneously orin sequential administration in a therapeutic or prophylactic treatmentmethod of the invention.

Also, an immune response is optionally modified or enhanced by, e.g.,administering one or more nucleic acids encoding one or more novelCD28BPs (or fragments thereof, including, e.g., soluble CD28BPs orfusion proteins thereof) or CTLA-4BPs (or fragments thereof, including,e.g., soluble CTLA-4BPs or fusion proteins thereof) with an antigen.Alternatively, an antigen response is optionally enhanced or modified byadministration of one or more CD28BPs (or fragments thereof, including,e.g., soluble CD28BPs) or CTLA4-BPs (or fragments thereof, including,e.g., soluble CTLA-4BPs) with an antigen.

CD28BPs and CTLA-4BPs, and B7-1 and B7-2 polypeptide variants describedherein (and nucleic acids encoding any of these polypeptides orfragments thereof), are useful in modulating the immune response in vivoin a variety of animals, (e.g., mammals, (including humans)) and invitro. These molecules are particularly useful in therapeutic and/orprophylactic applications when modulation of T cell responses isdesired. Examples of useful applications for CD28BP and/or CTLA4BP (orfragments thereof of each, or soluble and/or fusion protein versions ofeach) include conditions or diseases that may benefit from enhanced Tcell responses or where enhanced T cell responses are desired. They arealso useful, for example, in treating diseases where inhibition of Tcell proliferation/activation is desired. Examples of medical conditionsand/or diseases where enhanced T cell response is desired (e.g., by useof CD28BPs) (or fragments thereof, soluble and/or fusion proteinversions) include, for example, cancer, chronic infectious diseases, andvaccinations. Cancers include, but are not limited to, e.g., colorectalcancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer,naso-pharyngeal cancer, cancer, brain cancer, leukemia, melanoma, head-and neck cancer, stomach cancer, cervical cancer, ovarian cancer, andlymphomas.

CD28BPs, CTLA-4BPs, and B7-1 and B7-2 polypeptide variants describedherein (and nucleic acids encoding any of these), are useful in avariety of therapeutic and prophylactic treatment of diseases andconditions, including, e.g., allergy/asthma, neurological, organtransplantation (e.g., graft versus host disease, and autoimmunediseases), malignant diseases, chronic infectious diseases, including,but not limited to, e.g., viral infectious diseases, such as thoseassociated with, but not limited to, e.g., hepatitis B virus (HBV),herpes simplex virus (HSV), hepatitis C virus (HCV), HIV, humanpapilloma virus (HPV), and the like, and bacterial infectious diseases,such as, but not limited to, e.g., Lyme disease, tuberculosis, andchlamydia infections, and the like.

Furthermore, CD28BPs, CTLA-4BPs, and B7-1 and B7-2 polypeptide variantsdescribed herein (and nucleic acids encoding these) are useful inmethods for modulating production of specific cytokines, including thosediscussed in the Examples below. These molecules are particularly usefulin therapeutic and/or prophylactic applications in which an adjustment,alteration of a cytokine level, or production or stimulation of aspecific cytokine production is desired.

CD28BP polypeptides of the invention, or fragments thereof or solubleand/or fusion proteins thereof, modulate T cell proliferation oractivation and augment the immune response. In one embodiment, such aCD28BP polypeptide can be delivered in a treatment protocol as acomponent of a DNA vaccine vector, as a full-length polypeptide, as asoluble polypeptide subsequence of the full-length CD28BP polypeptide(e.g., ECD) used, if desired, as a polypeptide or protein vaccine or“boosting” polypeptide, or as a soluble fusion protein comprising afull-length CD28BP polypeptide or subsequence thereof, such as a solublepolypeptide subsequence (e.g., ECD); in such formats, the CD28BPpolypeptide may act as an agonist. In another embodiment, such as agenetic vaccine, in combination with a nucleic acid sequence encoding aspecific antigen, a nucleic acid sequence encoding a CD28BP polypeptideaugments the antigen specific T cell response for infectious disease orcancer antigens.

The CTLA-4BPs or fragments thereof or soluble and/or fusion proteinsthereof, of the invention can modulate T cell proliferation and/oractivation and inhibit the immune response in autoimmune diseases or, assoluble molecules, act as antagonists. Such a CTLA-4BP polypeptide canbe delivered in a treatment protocol as a component of a DNA vaccinevector, as a full-length polypeptide, as a soluble polypeptidesubsequence of the full-length CTLA-4BP polypeptide (e.g., ECD) used, ifdesired, as a polypeptide or protein vaccine or “boosting” polypeptide,or as a soluble fusion protein comprising a full-length CTLA-4BPpolypeptide or subsequence thereof, such as a soluble polypeptidesubsequence (e.g., ECD); in such formats, the CTLA-4BP polypeptide mayserve as an agonist.

As discussed above, genetic vaccine comprising a vector comprising anucleic acid sequence encoding a CTLA4-BP polypeptide and at least onenucleic acid sequence encoding at least one additional polypeptide ofinterest is also a feature of the invention. For example, in a DNAvaccine, in combination with a specific allergen, the CTLA-4BPs (orfragments thereof, or soluble and/or fusion proteins thereof) mayinhibit the allergen specific T cell response in allergy. Similarly, incombination with a specific auto-antigen, such as myelin basic protein,the CTLA-4BPs (or fragments thereof, or soluble and/or fusion proteinsthereof) may inhibit the auto-antigen-specific T cell response inautoimmunity, such as in multiple sclerosis.

Other clinical applications in which inducing tolerance is important andthus in which CTLA-4BPs are of use include autoimmunity (e.g., multiplesclerosis, rheumatoid arthritis, psoriasis), severe allergy and asthma,organ transplantation, generation of transgenic tissues to enablexenotransplants, graft versus host disease, and with components of genetherapy vectors to prolong survival of cells expressing foreignproteins.

Examples of medical conditions and/or diseases where down-regulation, orother altered type of T cell function by delivery of a CTLA-4BP of theinvention either as a DNA expression vector comprising a nucleic acidsequence encoding a CTLA-4BP polypeptide or as a soluble describedherein), or fragments thereof or soluble and/or fusion proteins thereof,may be of benefit include allergy, undesired immune response, autoimmunediseases, septic shock, and organ transplantation.

Examples of useful pathogen antigens, cancer antigens, allergens andauto-antigens for use in methods of the invention and/or in combinationwith NCSM polypeptides have been provided in the following commonlyassigned patent applications: Punnonen et al. (1999) WO 99/41369;Punnonen et al. (1999) WO 99/41383; Punnonen et al. (1999) WO 99/41368;and Punnonen et al. (1999) WO 99/41402), each of which is incorporatedherein by reference in its entirety for all purposes. Several otheruseful antigens have been described in the literature or can bediscovered using genomics approaches. Since typical tumor antigens areself proteins and thus host tolerant, it is optionally necessary togenerate “non-self” tumor antigens that induce cross-reactivity againstself tumor antigens also. This is optionally accomplished through, e.g.,recursive sequence recombination of existing tumor antigens from diversespecies to produce chimeric tumor antigens. Such chimeric antigens arethen screened for ones which activate antigen-specific T cells whichalso recognize the wild-type tumor antigen. Optional screenings testwhether chimeric antigens activate patient T cells (e.g., T cell linesspecific for wild-type antigens generated and activation induced by APCsexpressing recursively recombined antigens analyzed) and whether thechimeric antigen induces T cells that recognize wild-type antigen (e.g.,T cell lines specific for recursively recombined antigens generated andactivation induced by APCs expressing WT antigen analyzed).

A NCSM polypeptide of the invention is also useful in therapeutic orprophylactic treatment methods for treating or preventing any of theabove-mentioned diseases and disorders when administered to a subject asa polypeptide (e.g., administer at least one full-length or soluble NCSMor fragment thereof) or cell-based vaccine (e.g., cell expressing orsecreting at least one NCSM polypeptide) or a gene-based therapeuticpolypeptide (i.e., polypeptide product expressed by a NCSM gene),wherein such NSMC polypeptides are delivered alone or co-administeredsimultaneously or subsequently with one or more of an antigen, anotherco-stimulatory molecule, or adjuvant. A NCSM molecule is also useful fortreating or preventing any of the above-mentioned diseases and disorderswhen administered to a subject as a genetic vaccine (e.g., DNA vaccine)in which at least one NCSM polynucleotide is administered alone or in aplasmid vector or gene therapy format (i.e., a vector encoding at leastone NCSM polypeptide). Or, if desired, at least one NCSM polynucleotideis co-administered with a second DNA vector encoding at least one of anantigen, co-stimulatory molecule, and/or adjuvant. Alternatively, ifdesired, a vector comprising at least one NCSM-encoding polynucleotidesequence and at least one of an antigen, co-stimulatory molecule, and/oradjuvant can be prepared and administered to a subject in a treatmentprotocol; in this instance, the at least one NCSM-encodingpolynucleotide is co-expressed with at least one antigen, co-stimulatorymolecule, and/or adjuvant.

In another aspect, a soluble NCSM polypeptide may be used in methods fortreating or preventing any of the above-mentioned diseases or disorderswhen administered to a subject alone or in conjunction simultaneously orsubsequently with one or more of an antigen, another co-stimulatory,and/or adjuvant. The soluble NCSM may comprise a NCSM-ECD or subsequencethereof or may be formulated as a soluble fusion protein. Further, aNCSM polynucleotide that encodes a NSCM or any soluble form of a NCSM asdescribed herein is useful in therapeutic or prophylactic treatmentmethods for treating or preventing any of the above-mentioned diseasesor disorders when administered to a subject alone or in conjunction(simultaneously or subsequently) with one or more nucleotide sequencesencoding one or more of an antigen, another co-stimulatory, and/oradjuvant. If desired, the NCSM polynucleotide sequence and one or morepolynucleotide sequences encoding one or more of an antigen, anotherco-stimulatory, and/or adjuvant may be incorporated into one vector fordelivery to the subject and co-expression of the resulting NCSMpolypeptide, antigen, another co-stimulatory, and/or adjuvant.

As noted above, a variety of antigens can be delivered simultaneouslywith or following delivery of a NCSM molecule, where the NCSM moleculeis administered to the subject in either polypeptide or nucleic acidformat. The antigen may be delivered as a polypeptide or polynucleotide(via a vector). Antigens may be WT antigens or recombinant or chimericantigens, including, e.g., shuffled or mutated antigens.

Examples of cancer antigens that be used with NCSM molecules and inmethods of the invention described herein include, e.g., EpCam/KSA,bullous pemphigoid antigen 2, prostate mucin antigen (PMA) (Beckett andWright (1995) Int. J. Cancer 62:703–710), tumor associatedThomsen-Friedenreich antigen (Dahlenborg et al. (1997) Int. J. Cancer70:63–71), prostate-specific antigen (PSA) (Dannull and Belldegrun(1997) Br. J. Urol. 1:97–103), luminal epithelial antigen (LEA.135) ofbreast carcinoma and bladder transitional cell carcinoma (TCC) (Jones etal. (1997) Anticancer Res. 17:685–687), cancer-associated serum antigen(CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al. (1995)Gynecol. Oncol. 59:251–254), the epithelial glycoprotein 40 (EGP40)(Kievit et al. (1997) Intl. J. Cancer 71:237–245), squamous cellcarcinoma antigen (SCC) (Lozza et al. (1997) Anticancer Res. 17:525–529), cathepsin E (Mota et al. (1997) Am. J. Pathol. 150:1223–1229),tyrosinase in melanoma (Fishman et al. (1997) Cancer 79: 1461–1464),cell nuclear antigen (PCNA) of cerebral cavernomas (Notelet et al.(1997) Surg. Neurol. 47: 364–370), DF3/MUC1 breast cancer antigen(Apostolopoulos et al. (1996) Immunol. Cell. Biol. 74: 457–464; Pandeyet al. (1995) Cancer Res. 55: 4000–4003), carcinoembryonic antigen(Paone et al. (1996) J. Cancer Res. Clin. Oncol. 122:499–503; Schlom etal. (1996) Breast Cancer Res. Treat. 38:27–39), tumor-associated antigenCA 19-9 (Tolliver and O'Brien (1997) South Med. J. 90:89–90; Tsuruta etal. (1997) Urol. Intl. 58:20–24), human melanoma antigensMART-1/Melan-A27-35 and gp100 (Kawakami and Rosenberg (1997) Intl. Rev.Immunol. 14:173–192; Zajac et al. (1997) Intl. J. Cancer 71:491–496),the T and Tn pancarcinoma (CA) glycopeptide epitopes (Springer (1995)Crit. Rev. Oncog. 6:57–85), a 35 kD tumor-associated autoantigen inpapillary thyroid carcinoma (Lucas et al. (1996) Anticancer Res.16:2493–2496), KH-1 adenocarcinoma antigen (Deshpande and Danishefsky(1997) Nature 387:164–166), the A60 mycobacterial antigen (Maes et al.(1996) J. Cancer Res. Clin. Oncol. 122:296–300), heat shock proteins(HSPs) (Blachere and Srivastava (1995) Semin. Cancer Biol. 6:349–355),and MAGE, tyrosinase, melan-A and gp75 and mutant oncogene products(e.g., p53, ras, CDk4, and HER-2/neu (Bueler and Mulligan (1996) Mol.Med. 2:545–555; Lewis and Houghton (1995) Semin. Cancer Biol. 6:321–327; Theobald et al. (1995) Proc. Nat'l. Acad. Sci. USA 92:11993–11997), prostate specific membrane antigen (PSMA) Bangma CH et al.(2000) Microsc Res Tech 51:430–5, TAG-72, McGuinness RP et al. Hum GeneTher (1999) 10:165–73, and variants, derivatives, and mutated, andrecombinant forms (e.g., shuffled forms) thereof of these antigens.

To generate, e.g., vaccines with evolved NCSM molecules, pre-clinicalstudies can first be done in, e.g., mice. Mice can be used for study of,e.g., CTLA-4BPs because, e.g., effects of the protein are more difficultto study in vitro, work in monkeys is less cost effective than work withmice, mouse models of autoimmune diseases have been established givingexcellent means to study induction and breaking tolerance, and the samemouse models can be used for biological characterization of CD28BPs aswell. Pre-clinical mice studies can allow optimization of, e.g., vectorsfor specific targets of interest, pharmacokinetics, drug half life,adjuvant stability and in vivo efficacy of such things as DNA vaccinesand soluble protein administration. Mouse studies optionally can befollowed by pre-clinical studies in non-human primates. Non-humanprimate trials can, e.g., optimize efficacy in boosting the innateimmune system as well as optimize efficacy of use of NCSM molecules asvaccine adjuvants. Non-human primate trials optionally can serve to,e.g., optimize protective immunity and thereby help identify the bestvaccine for human clinical trials.

As an illustrative, but not limiting example, either or both types ofNCSM (i.e., CD28BPs and CTLA-4BPs) or fragments thereof can be used in aboosting method or format to modify an immune response. This methodtypically comprises, e.g., initially administering a DNA vaccine (a“prime boost”) to a subject, followed by, e.g., a second administrationwith, e.g., one or more of the NCSM polypeptide molecules either inpolypeptide format or in nucleic acid format.

Furthermore, the invention also provides for gene therapy vectorscomprising at least one nucleotide sequence encoding at least oneCTLA-4BP or fragment, variant or homologue thereof. In one aspect, agene therapy vector (e.g., adenovirus (AV), adeno-associated virus(AAV), retrovirus, poxvirus, or lentivirus vectors) comprising at leastone nucleic acid sequence encoding at least one CTLA-4BP polypeptide orfragment thereof) is used to reduce recognition of the transduced cellsby specific T cells. The incorporation of the CTLA-4BP-encoding nucleicacid sequence helps to prolong survival of the gene therapy vector. Ingene therapy, when the therapeutic or prophylactic transgene isexpressed by the host cells, these cells are often also recognized bythe cells of the immune system and cytotoxic T cells may destroy thecells expressing the transgene, thereby limiting the efficacy of genetherapy. If the cells expressing the transgene simultaneously express aCTLA-4BP, this will reduce the activity of those cytotoxic T cells,thereby prolonging the survival of the transduced cells and improvingthe efficacy of gene therapy.

The present invention additionally provides a method to design oridentify small molecule agonists and antagonists that either enhance orinhibit signaling through CD28 and/or CTLA-4 molecules. Methods known tothose skilled in the art, such as X-ray crystallography, are used toidentify the 3-dimensional structures of proteins (i.e., the CD28BPs andCTLA-4BPs of the invention) and fragments thereof of each. These andother methods can be used to identify and determine the conformationsand structures that contribute to the preferential binding of the NMCSmolecules (e.g., CD28BPs and CTLA-4BPs) of the invention to CD28 andCTLA-4. Based on the information obtained, small molecules thatspecifically bind to CD28 or CTLA-4 can be designed. Functionalscreening assays known to those skilled in the art, such as in vitro Tcell proliferation/activation assays, can be used to analyze whethersuch molecules are specific antagonists or agonists. The resulting smallmolecules that are agonists for CD28 and/or antagonists for CTLA-4 canbe used to, e.g., enhance or modify T cell dependent immune responses.Similarly, small molecules that are antagonists for CD28 and/or agonistsfor CTLA-4 can be used to, e.g., down-regulate or modify T cell specificimmune responses and/or to induce tolerance and/or anergy. These varioustypes of small molecules optionally are beneficial as, e.g., vaccineadjuvants and, e.g., in treating diseases when manipulation of T cellresponse is desired.

The invention includes methods of designing or identifying CD28 agoniststhat enhance or inhibit signaling through either CD28 or CTLA-4molecules of T-cells, based on visual viewing and/or analysis of thethree-dimensional structure (e.g., X-ray crystallography), an analysisof the residues involved in CD28 and/or CTLA-4 binding, and thepositions and types of such residues of any of the polypeptides of theinvention as found in SEQ ID NOS:48–94, 174–252, 263–272, 283–293, orfragments thereof.

The invention also includes methods of treating a disease or disorder ina subject in need of such treatment, comprising: administering to thesubject any NCSM polypeptide described herein in an amount effective totreat said disease or disorder. In another aspect, the inventionprovides methods for therapeutic or prophylactic treatment of a diseaseor disorder in a subject in need of such treatment, comprising:administering to the subject any NCSM polypeptide and an immunogenspecific for said disease or disorder, wherein the combined amount ofpolypeptide and immunogen is effective to prophylactically ortherapeutically treat said disease or disorder. In some such methods,the polypeptide is present in an amount sufficient to enhance, diminishor modify an immune response induced by the immunogen. The compositionmay comprise the polypeptide, the immunogen, and a pharmaceuticallyacceptable excipient is administered to the subject in an amounteffective to treat the disease or disorder. For all such methods, thesubject may be a mammal, including, e.g. a human. Further, for some suchmethods, the polypeptide is administered in vivo to the subject or exvivo to a population of cells obtained from the subject. In anotheraspect, the invention includes methods for treating a disease ordisorder described herein in a subject in need of such treatment,comprising administering to the subject a NCSM polypeptide in an amounteffective to treat the disease or disorder.

The invention includes methods of designing or identifying CD28 agoniststhat enhance or inhibit signaling through either CD28 or CTLA-4molecules of T-cells, based on visual viewing and/or analysis of thethree-dimensional structure (e.g., X-ray crystallography), an analysisof the residues involved in CD28 and/or CTLA-4 binding, and thepositions and types of such residues of any of the polypeptides of theinvention as found in SEQ ID NOS:48–94, 174–252, 263–272, 283–293, orfragments thereof.

The NCSM polynucleotides and polypeptides, B7-1 and B7-2 polynucleotidevariants and polypeptide variants, and fragments and homologues of allsuch molecules as described throughout, can be administered to a subjectby any one of the delivery routes described below (including, but notlimited to, e.g., intramuscularly, intradermally, subdermally,subcutaneously, orally, intraperitoneally, intrathecally, intravenously,mucosally, systemically, parenterally, via inhalation, or placed withina cavity of the body (including, e.g., during surgery)).

Integrated Systems

The present invention provides computers, computer readable media, andintegrated systems comprising character strings corresponding to thesequence information herein for the polypeptides and nucleic acidsherein, including, e.g., those sequences listed herein and varioussilent substitutions and conservative substitutions thereof. Variousmethods and genetic algorithms (GAs) known in the art can be used todetect homology or similarity between different character strings, orcan be used to perform other desirable functions such as to controloutput files, provide the basis for making presentations of informationincluding the sequences and the like. Examples include BLAST, discussedsupra.

Thus, different types of homology and similarity of various stringencyand length can be detected and recognized in the integrated systemsherein. For example, many homology determination methods have beendesigned for comparative analysis of sequences of biopolymers, forspell-checking in word processing, and for data retrieval from variousdatabases. With an understanding of double-helix pair-wise complementinteractions among 4 principal nucleobases in natural polynucleotides,models that simulate annealing of complementary homologouspolynucleotide strings can also be used as a foundation of sequencealignment or other operations typically performed on the characterstrings corresponding to the sequences herein (e.g., word-processingmanipulations, construction of figures comprising sequence orsubsequence character strings, output tables, etc.). An example of asoftware package with GAs for calculating sequence similarity is BLAST,which can be adapted to the present invention by inputting characterstrings corresponding to the sequences herein.

Similarly, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can be adapted to the present invention by inputting acharacter string corresponding to the NCSM polypeptides orpolynucleotides of the invention or both, or fragments of either. Forexample, the integrated systems can include the foregoing softwarehaving the appropriate character string information, e.g., used inconjunction with a user interface (e.g., a GUI in a standard operatingsystem such as a Windows, Macintosh or LINUX system) to manipulatestrings of characters. As noted, specialized alignment programs such asBLAST can also be incorporated into the systems of the invention foralignment of nucleic acids or proteins (or corresponding characterstrings).

Integrated systems for analysis in the present invention typicallyinclude a digital computer with GA software for aligning sequences, aswell as data sets entered into the software system comprising any of thesequences described herein. The computer can be, e.g., a PC (Intel x86or Pentium chip-compatible DOS™, OS2™ WINDOWS™ WINDOWS NT™, WINDOWS95™,WINDOWS98™ LINUX based machine, a MACINTOSH™, Power PC, or a UNIX based(e.g., SUN™ work station) machine) or other commercially common computerwhich is known to one of skill. Software for aligning or otherwisemanipulating sequences is available, or can easily be constructed by oneof skill using a standard programming language such as Visualbasic,Fortran, Basic, Java, or the like.

Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation.

The software can also include output elements for controlling nucleicacid synthesis (e.g., based upon a sequence or an alignment of asequence herein) or other operations which occur downstream from analignment or other operation performed using a character stringcorresponding to a sequence herein.

The invention provides a computer or computer readable medium comprisinga database comprising a sequence record comprising one or more characterstring corresponding to a nucleic acid or protein sequence selected fromSEQ ID NOS:1–272 and 283–293.

In another aspect, the invention provides an integrated systemcomprising a computer or computer readable medium comprising a databasecomprising at least one sequence record, each comprising at least onecharacter string corresponding to a nucleic acid or protein sequenceselected from SEQ ID NOS:1–272 and 283–293, the integrated systemfurther comprising a user input interface allowing a user to selectivelyview one or more sequence records. For some such integrated systems, thecomputer or computer readable medium comprising an alignment instructionset which aligns the character strings with at least one additionalcharacter string corresponding to a nucleic acid or protein sequence.The instruction set may comprise one or more of: a local homologycomparison determination, a homology alignment determination, a searchfor similarity determination, and a BLAST determination. Some suchsystems may further comprise a user readable output element whichdisplays an alignment produced by the alignment instruction set.

In some aspects, the computer or computer readable medium furthercomprises an instruction set which translates at least one nucleic acidsequence comprising a sequence selected from SEQ ID NOS:1–47, 95–173,and 253–262 into an amino acid sequence. In other aspects, the computeror computer readable medium further comprising an instruction set forreverse-translating at least one amino acid sequence comprising asequence selected from SEQ ID NOS:48–94, 174–252, 263–272, and 283–293,into a nucleic acid sequence. For some such systems, the instruction setselects the nucleic acid sequence by applying a codon usage instructionset or an instruction set which determines sequence identity to a testnucleic acid sequence.

Also provided is a method of using a computer system to presentinformation pertaining to at least one of a plurality of sequencerecords stored in a database, each of said sequence records eachcomprising at least one character string corresponding to SEQ IDNOS:1–272 and 283–293, the method comprising: determining a list of oneor more character strings corresponding to one or more of SEQ IDNOS:1–272 and 283–293, or a subsequence thereof; determining which oneore more character strings of said list are selected by a user; anddisplaying the selected character strings, or aligning the selectedcharacter strings with an additional character string. Some such methodsfurther comprise displaying an alignment of the selected characterstring with the additional character string and/or displaying the list.

Kits

The present invention also provides kits including the NCSMpolypeptides, polynucleotides, expression vectors, cells, vaccines,methods, compositions, systems, and apparatuses of the invention. Kitsof the invention optionally comprise at least one of the following ofthe invention: (1) an apparatus, system, system component, or apparatuscomponent as described herein; (2) at least one kit component comprisinga NCSM polypeptide or polynucleotide, soluble NCSM polypeptide orpolynucleotide, or fragment thereof; an NCSM-Ig or NCSM-ECD-Ig fusionprotein; plasmid expression vector encoding a NCSM polypeptide, solubleNCSM polypeptide, or fragment thereof; cell expressing a NCSMpolypeptide, soluble NCSM polypeptide, or fragment thereof; acomposition or vaccine composition comprising at least one of any suchcomponent; (3) instructions for practicing any method described herein,including a therapeutic or prophylactic methods, instructions for usingany component identified in (2) or any vaccine or composition of anysuch component; and/or instructions for operating any apparatus, systemor component described herein; (4) a container for holding said at leastone such component or composition, and (5) packaging materials.

In a further aspect, the present invention provides for the use of anyapparatus, component, composition, or kit described above and herein,for the practice of any method or assay described herein, and/or for theuse of any apparatus, component, composition, or kit to practice anyassay or method described herein.

EXAMPLES

The following examples are offered to illustrate the present invention,but not to limit the spirit or scope of the present invention in anyway.

Materials and Methods:

A. Isolation of Mammalian Parental cDNAs for Library Construction.

Human, rhesus monkey, baboon, orangutan, cow (GenBank Ace. No. Y09950),cat, and rabbit (GenBank Acc. No. D49843) wild-type B7-1 (CD80) parentalgenes were cloned by the reverse transcriptase polymerase chain reaction(RT-PCR) method. RAJI, PUTI, LCL8664, and 26CB-1 cell lines were used assources of total or messenger RNA (mRNA) for human (Homo sapiens),orangutan (Pongo pygmaeous), rhesus monkey (Macaca mulatta)(GenBank Acc.No. U19840) and baboon (Papio hamadryas) B7-1 genes for B7-1 cDNApreparation. mRNA or total RNA encoding feline (Felis catus), bovine(Bos taurus) and rabbit (Oryctolagus cuniculus sub-species domesticus)B7-1 genes for B7-1 cDNA preparation were obtained from peripheral bloodmononuclear cells (PBMCs) derived from the respective species. PBMCswere isolated from cat, cow, and rabbit intravenous blood draws byFicoll gradient separation. The cells were then activated for 2 days inDulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum (HyClone, Logan, Utah), 5 microgram/milliliter (ug/ml)lipopolysaccharides (LPS), 0.25 μg/ml pokeweed mitogen (PWM) and 0.1μg/ml phytohemagglutinin (PHA). Cell lines were maintained at 37° C. inDMEM containing 10% fetal calf serum.

The cell lines or activated PBMCs were harvested and mRNA or total RNAwas isolated using FastTrack 2.0 mRNA isolation kit (Invitrogen,Carlsbad, Calif.) or Promega RNAgents Total RNA Isolation System kit(Promega, Madison, Wis.), respectively. Primers used to clone therespective mammalian B7-1 cDNAs were designed based on publishedsequences for human, bovine and rabbit B7-1 genes (see, e.g., Freeman,G. J. et al. (1989) J Immunol 143:2714–22; Parsons, K. R. & Howard, C.J. (1999) Immunogenetics 49:231–4; and Isono, T. & Seto, A. (1995)Immunogenetics 42:217–20)(see also, for human B7-1, GenBank Access. Nos.U33208, AF024703; Cow B7-1, GenBank Acc. No. Y09950; rabbit B7-1,GenBank Access. No. D49843. The primers, which were purchased from GibcoBRL, contained a Bam H I site 5′ of the start codon and a Kpn I site 3′of the stop codons. cDNA was generated using the mRNA or total RNA inthe Invitrogen cDNA Cycle kit. The cDNAs were generated by RT-PCR, whichwas performed using the cDNA Cycle kit (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions and standard techniques.Each cDNA was then used as a template for PCR generation of, e.g.,double-stranded cDNA using primer(s) specific for each species.

All primers used to clone B&-1 (CD80) cDNAs contained a Bam HI site 5′of the species start codon and a Kpn I site 3′ of the species stopcodons. The PCR products were gel purified using the Bio101 GENECLEAN IIkit, digested with BamHI and Kpn I or Asp 718, and ligated into apcDNA3.1 (−) expression vector (Invitrogen, Carlsbad, Calif.) digestedwith BamHI and Kpn I or Asp 718. The Life Technologies E. coli strainDH10B was transformed with the cDNA clones; colonies were picked, grown,and the clones were isolated from the bacteria using the Qiagen PlasmidMaxi kit (Qiagen, Valencia, Calif.).

B. Generation of Recombinant Nucleic Acid Libraries.

Recombinant libraries comprising recombinant (chimeric) nucleic acidsequences were generated by recursive sequence recombination proceduresusing the seven mammalian cDNAs isolated as described above. In oneaspect, libraries comprising shuffled chimeric nucleic acid sequenceswere generated by applying DNA shuffling procedures to the sevenmammalian cDNA sequences as described previously in, e.g., Stemmer, W.(1994) Nature 370:389–391 (1994) and Crameri, A. et al. (1998) Nature391:288–91, and other references cited above in the section describingrecursive sequence recombination and shuffling methods. The shufflednucleotide sequences were digested with Bam HI and Asp 718 and gelpurified using standard techniques. The resulting chimeric shufflednucleotide sequences were cloned into a pcDNA3.1⁻ expression vector(Invitrogen, Carlsbad, Calif.) using standard cloning techniques (see,e.g., Sambrook, supra) and according to manufacturer's instructions. Thevector was used for transfecting cells as described below.

The FLAG sequence (DYKDDDDK (SEQ ID NO:296)) was inserted at thejunction separating the sequence encoding the signal peptide and thesequence encoding the mature polypeptide (e.g., mature coding region)for each of the human B7-1 and CD28BP clones (e.g., CD28BP-15) using theExSite PCR site-directed mutagenesis kit (Stratagene, San Diego, Calif.)according to manufacturer's instructions. The nucleotide sequencescorresponding to the signal sequence and mature coding region weredetermined for each shuffled nucleotide sequence by comparison with theknown sequences corresponding to the signal sequence and mature codingregion for hB7-1. Mutagenesis primers were designed with the FLAGsequence flanked by 24 nucleotides of signal and mature coding sequencespecific to each clone. Plasmid DNA was prepared and purified from thecDNA libraries following standard procedure in Maniatis et al.,Molecular Cloning: A laboratory Manual, Cold Spring Harbor, N.Y. (1987).

C. Protein Conjugation.

Soluble CD28-Ig (sCD28-Ig) is a soluble fusion protein between theextracellular domain of human CD28 and the Fc portion of humanimmunoglobulin G (IgG); soluble CTLA-4-Ig (sCTLA-4-Ig) is a solublefusion protein between the extracellular domain of human CTLA-4 and ahuman Ig C gamma chain (see, e.g., Linsley et al. (1991), J Exp Med174:561–569). Soluble CD28-Ig Fc (Fc portion of human IgG1) and solubleCTLA-4-Ig Fc (Fc portion of human IgG1) fusion proteins were bothobtained from R&D Systems, Minneapolis, Minn. NHS-biotin was obtainedfrom Pierce (Rockford, Ill.) and Fluorescein Isothiocyanate Isomer I(FITC) was obtained from Molecular Probes (Eugene, Oreg.).

Molar ratios of 1:38.5 for CTLA-4-Ig Fc:FITC and 1:35 for CD28-IgFc:Biotin were used during the conjugation. Proteins at 1–3 mg/ml weredialyzed vs. 0.1 M Carbonate buffer for FITC conjugation and 0.1M SodiumBicarbonate buffer for Biotin conjugation. Ratios of 155 ug FITC/1 mg ofCTLA-4-Ig Fc and 124 ug Biotin/1 mg CD28Fc were used during theconjugation. FITC or biotin at 2 mg/ml in dimethyl sulfoxide (DMSO) wasadded dropwise while vortexing to dialyzed protein, incubated at 25° C.in the dark for 2 hours, and then dialyzed against PBS overnight toexchange buffers. (For additional methods, see Linsley et al., supra.)

D. Binding Activity Assays and Flow Cytometry.

Soluble CD28-Ig and soluble CTLA-4-Ig fusion proteins were conjugatedwith biotin and fluorescein isothiocyanate, respectively, as describedabove. The transfectants were treated with 0.5 mM EDTA in PBS/2% FCS for15–30 minutes. Several representative wells were counted to determinethe number of cells per well. An equal volume of conjugated sCD28-Ig(Fc) or conjugated sCTLA-4-Ig (Fc) was added to the transfected 293 orCOS-7 cells at appropriate concentrations as follows. For competitivebinding assays, the transfected cells were first incubated withbiotin-conjugated sCD28-Ig at room temperature. FITC-conjugatedCTLA-4-Ig was then added to this incubation mixture 15 minutes (min)after the addition of biotin-conjugated sCD28-Ig, and the entire mixturewas incubated for an additional 1 hour and 45 minutes. (Alternatively,an individual binding assay can be performed using the same procedure,but in which the transfected cells are incubated individually withbiotin-conjugated sCD28-Ig or FITC-conjugated CTLA-4-Ig.)

The labeled cells were subsequently handled at 4° C., washed twice withDMEM/10% FCS and incubated with 0.1 μg/ml Streptavidin-phycoerythrin(PE) (Pharmingen, San Diego, Calif.) in 100 μl for 15 min. Biotin bindsthe fluorescence marker Streptavidin-PE. (R-PE has an excitation maximumof 565 nanometers (nm) and an emission maximum of 578 nm. B-PE hasexcitation and emission maxima of 545 nm and 578 nm, respectively. FITChas excitation and emission maxima of 494 and 519, respectively.) Thecells were again washed twice and resuspended in 200 μl medium with 5μg/ml propidium iodide (PI). The cells were then analyzed using aFACSCalibur flow cytometer and CellQuest software (BDIS, San Jose,Calif.). Cell sorting was performed by flow-cytometry based cell sortingscreening methods using FACS (Fluorescence-Activated Cell Sorting)Vantage SE cell sorter (BDIS) (Becton Dickinson; San Jose, Calif.). Thestaining concentration was determined for each labeled protein toprovide a maximal Mean Fluorescence Intensity (MFI) and minimalbackground signal (e.g., optimum staining concentration was theconcentration per 10⁶ cells).

Representative binding profile for the competitive binding assays areshown in FIGS. 4A–4D. For the individual binding assay, individualbinding profiles are generated for binding of the transfected cells toeach of the biotin-conjugated sCD28-Ig or FITC-conjugated CTLA-4-Ig(data not shown).

For a description of flow cytometry cell sorting methods, which areknown in the art, see Current Protocols in Immunology, John Colligan etal., eds., Vols. I–IV (John Wiley & Sons, Inc., 1991 and 2001Supplement); Sambrook; Rapley and Walker, all supra, each of which isincorporated herein by reference in its entirety for all purposes.

E. Library Sorting/Enrichment.

Libraries were pre-enriched by FACS sorting for preferential binding toCTLA-4 over CD28 or for preferential binding to CD28 over CTLA-4.Library sorting/enrichment was performed as follows. 293 cells weretransfected with a bulk population of recombinant clones from therecombinant libraries, and each transfected library was incubated withboth soluble reagents sCD28-Ig and sCTLA-4-Ig (see Section D, above).Transfectants from the CTLA-4-Ig binding biased library were incubatedwith an optimal concentration of sCD28-Ig and a 10-fold lowerconcentration of sCTLA-4-Ig than optimal. Transfectants from the CD28-Igbinding biased library were labeled with optimal amounts of both solublereagents. Cells that preferentially bound CD28 over CTLA-4 were sortedfrom the CD28-Ig binding biased library transfectants, and cells thatpreferentially bound CTLA-4 over CD28 were sorted from the CTLA-4-Igbinding biased library transfectants.

Plasmid was recovered from the sorted cells by lysis with 400 μl Hirt'ssolution (0.6% sodium dodecyl sulfate (SDS), 10 milliMolar (mM) EDTA pH8.0) for 0.5 hour, the addition of 100 μl of 0.5 M NaCl to the lysate,and the lysate incubated over night. The lysate was spun (e.g.,centrifuged at 14,000×g for 60 minutes), extracted with equal volume ofPhenol/Chloroform, ethanol precipitated, and resuspended in 10 μl TEbuffer. The isolated plasmid was used to transform E. coli strain DH10Band the transformed cells were plated on LB agar plates. All colonieswere harvested and combined and plasmid DNA was isolated using theQiagen Maxiprep kit.

F. DNA Purification and Transfections.

E. coli strain DH10B (Life Technologies, Rockville, Md.) was transformedwith Maxiprep DNA from the libraries comprising recombinant (chimeric)nucleic acid clones generated by DNA shuffling or other recursivesequence recombination procedures, as described above. The transformedcells were plated overnight. Individual colonies were picked from theplated libraries and inoculated into 96-well blocks containing 1.2 mlTerrific Broth-amp (50 μg/ml). Each block was also inoculated with apcDNA3.1- expression vector (Invitrogen, Carlsbad, Calif.) (controlvector) and human CD80 each in one well. The 96-well plate cultures weregrown for 20 hours at 37° C., and plasmid DNA was purified using theBiorobot (Qiagen, Valencia, Calif.). Cells of either the mammalian cellline 293 or COS-7 (or other cell line f=of interest) were plated in96-well plates at a density of 2×10⁴ cells per well the day prior totransfections. The cells were transfected with plasmids encoding awild-type B7-1 or chimeric polypeptide using Superfect (Qiagen) orLipofectamine (Life Technologies) according to the manufacturer'sinstructions.

The following procedure was used for large-scale transfections.Large-scale transfections are typically used in pre-enrichment sorts,and for the generation of therapeutic and/or prophylactic tumorvaccines, including, e.g., composition comprising NCSMpolypeptide-expressing tumor cells. Human embryonic kidney 293 cells (oralternatively, e.g., monkey COS-7 cells or tumor cells) were transfectedwith human CD80 (B7-1) plasmid DNA using Life Technologies Lipofectamineand OptiMEM medium. Per 20 cm² of plated 293 cells, 3 micrograms (μg)DNA in 200 microliters (μl) OptiMEM were combined with 18 μlLipofectamine in 200 μl OptiMEM. This mixture was incubated for 15–30minutes at 25° C., 1.6 milliliters (ml) OptiMEM were added, and 2 ml ofthis mixture were added per 20 cm² of plated 293 cells. Transfectionswere performed in T25 to T175 flasks containing 60–80% confluent 293.Cells were incubated for 5–7 hours in a 37° C. humidified incubatorcontaining 5% CO₂. An equal volume of Dulbecco's modified Eagle's medium(DMEM)/20% fetal calf serum (FCS) (HyClone, Logan, Utah) was added tothe flask and incubated overnight. Cells were trypsinized, replated, andincubated for 24 hours. Cells were then removed from plastic using EDTAtreatment. Cells transfected with, respectively, a control vector,pcDNA3.1−expression vector (Invitrogen, Carlsbad, Calif.), or plasmidvector encoding human CD80 (B7-1) were counted and aliquoted at 2×10⁶cells/ml.

The following procedure was used for high-throughput (HTP) transfectionsfor screening library clones in both T cell and binding assays. ForLipofectamine HTP transfections, plated 293 or COS-7 cells were washed1× with 200 μl PBS, and 50 μl OptiMEM was added to each well. DNA cloneconcentrations were normalized to 100 ng/μl±33 ng/ul. The DNApreparation was diluted to 5 ng/μl±33% in OptiMEM, and 50 μl was platedper well in empty 96-well U bottom plates. 50 μl of OptiMEM withLipofectamine at 0.03 ul/1 μl OptiMEM was added to each well containingdiluted DNA preparation. Each well contained 50 ng DNA±33% and 0.3 μlLipofectamine. The mix was incubated for 15 minutes at room temperature,and then 20 μl per well of the mixture was added to each well containing293 or COS-7 cells in 50 μl OptiMEM. The cells were incubated at 37° C.for 5–7 hours, 70 μl DMEM/20% FCS was added to each well and the plateswere incubated overnight. The wells were subsequently trypsinized,washed 2× with DMEM10% FCS, replated in sterile V-bottom plates, andincubated overnight. Alternatively, a Superfect (Qiagen) HTP protocolfor transfection, and like transfection protocols, can be used byfollowing the manufacturer's instructions.

G. T Cell Proliferation Assays.

Peripheral blood was obtained from healthy blood donors as standardbuffy coat preparations collected at Stanford Medical School BloodCenter (Palo Alto, Calif.). Peripheral blood mononuclear cells (PBMC)were isolated from human blood by centrifugation over Histopaque-1077(Sigma, St. Louis, Mo.) (using Ficol gradient separation).

T cells were isolated and purified either by staining the cells withanti-human CD2 monoclonal antibodies (mAbs) and sorting for CD2 positive(CD2⁺) cells using a FACS Vantage SE or removing cells that stained withmAbs specific for CD14, CD20, CD56 and CD94 by magnetic beads(Dynabeads, Dynal, Lake Success, N.Y.); Abs were purchased fromPharmigen (San Diego, Calif.). Magnetic separation of the T cells usingDynal Dynabeads was performed by first labeling PBMCs with puremonoclonal antibodies against CD 14, CD20, CD56 and CD94, then labelingthe cells with Sheep anti-mouse Dynabeads. Non-T cells were removed bydepleting with a magnet. The purity of the T cells was 96–99% whenanalyzed by staining with anti-CD3 mAbs purchased from Pharmigen (SanDiego, Calif.).

T cell proliferation was measured by ³H-thymidine incorporation.Briefly, 293 cells (or COS-7, or other cells of interest) weretransfected as described above for HTP transfections with a plasmid(expression vector) encoding a hB7-1 (or other mammalian B7-1), achimeric CD28BP or CTLA-4BP polypeptide (i.e., a clone selected from theCD28-Ig/CTLA-4-Ig binding screen assay), or with a control vectorlacking the B7-1 or NCSM nucleic acid insert. (The Effectine HTP(Qiagen, Valencia, Calif.) 96-well transfection method, which can alsobe used for plasmid transfections, was used to transfect cells withCTLA-4BP selected from Round 1 libraries according to the manufacturer'sinstructions.) Twenty-four hours after transfection, 5×10⁴ purified Tcells were cultured in triplicate in the presence of irradiated (5000rads) transfectants and soluble anti-human CD3 mAbs (5 μg/ml) U-bottom96-well plates (VWR, West Chester, Pa.) at 37° C. in a humidifiedatmosphere containing 5% CO₂ in Yssel's medium supplemented with 10% FCS(200 μl/well) for a total of 3 days (72 hours). 1 microCurie (μCi)/wellof ³H-thymidine (Amersham, Piscataway, N.J.) was added by pulsing to thecell cultures during the last 8 hours of the culture period, and thecells were harvested for counting onto filter paper by a cell harvester(Tomtec, Hamden, Conn.). Yssel's medium is described in Yssel et al.(1984) J Immunol Methods 72(1):219. ³H-thymidine uptake/incorporation inthe cultured cells was determined by measuring the radioactivity on thedried filters using a MicroBeta scintillation counter (Wallac, Turku,Finland). Proliferation of T cells is expressed as the mean counts perminute (cpm) of triplicate wells. The results shown are representativeof typically an average of 6 experiments. Chimeric clones that inducedor inhibited T cell proliferation at a level about equal to, greaterthan, or less than that observed with human CD80 (hB7-1) were identifiedand selected for further characterization.

H. Mixed Lymphocyte Culture Assay.

Proliferation of purified T cells was also measured in mixed lymphocytecultures (MLC). Mixed lymphocyte reaction (MLR) was performed usingirradiated PBMC as stimulator cells and allogeneic PBMC as responders.Stimulator cells were irradiated (2500 rads) and co-cultured withallogeneic PBMC (1×10⁵ cells/well) in 96-well flat-bottomed microtiterculture plates (VWR) at 1:1 ratio for a total of 5 days. During the last8 hours of the culture period, the cells were pulsed with 1 uCi/well of³H-thymidine, and the cells were harvested for counting onto filterpaper by a cell harvester as described above. ³H-thymidine incorporationwas measured as described above for purified T cells. Proliferation of Tcells was expressed as the mean cpm of triplicate wells. The resultsshown representative of more than one experiment.

I. Analysis of Cytokine Levels in Culture Supernatants.

Supernatants of cell cultures from mixed lymphocyte reaction werecollected after 48 hours and stored at −80° C. until they were analyzedfor the presence of various cytokines. IL-10 and IFN-gamma levels weredetermined in duplicate using cytokine-specific ELISA kits (R&D Systems,Minneapolis, Minn.) by following the manufacturer's instructions (R&DSystems, Minneapolis, Minn.). Controls were provided in the kits.

J. Concentration-Dependent CTLA4-Ig or CD28-Ig Binding Assays.

For each particular transfectant, 2×10⁵ cells were incubated with serialdilutions of CTLA-4-Ig in phosphate-buffered saline (PBS) containing 5%FCS for 30 minutes on ice. Next, the cells were washed with PBS-FCS andincubated subsequently with a saturating concentration ofFITC-conjugated goat anti-human IgG mAb (Fc specific) (CaltagLaboratories, San Francisco, Calif.) for another 30 minutes on ice. Thecells were analyzed using a FACSCalibur flow cytometer and CellQuestsoftware, and cell sorting was performed using FACS Vantage SE cellsorter (BDIS) described above. Plasmids were recovered from the sortedcells by Hirt preparation as described above.

Example I Cloning of Parent cDNA Sequences for Library Construction

The cDNAs encoding human, rhesus monkey, orangutan, baboon, bovine,rabbit, and feline B7-1 (CD80) co-stimulatory molecules were cloned byRT-PCR. These starting B7-1 genes encoded polypeptide molecules with, bycomparison, amino acid sequence identities ranging from about 58–98%amino acid sequence identity using Jotun Hein method, DNASTAR (inMegaLine™ DNASTAR package, MegaLine™ Ver. 4.03), followingmanufacturer's instructions and using default values specified in theprogram. The polynucleotide sequences for baboon B7-1 (SEQ ID NO:46) andorangutan B7-1 (SEQ ID NO:47) are examples of WT NCSM polynucleotideswhose sequences were previously unknown. These baboon B7-1 and orangutanB7-1 polynucleotides, as well as homologues and baboon B7-1 (SEQ IDNO:93) and orangutan B7-1 (SEQ ID NO:94) polypeptides encoded therefrom,are included as NCSM molecules of the invention.

The RAJI, PUTI, LCL8664, and 26CB-1 cell lines were used as sources ofmessenger or total RNA for primate B7-1 cDNA preparation as describedpreviously. Intravenous draws of peripheral blood were used as thesource of messenger or total RNA for cat, cow, and rabbit B7-1 cDNApreparation as discussed previously. Peripheral blood mononuclear cells(PBMCs) were isolated from the cat, cow, and rabbit blood draws by Ficolgradient separation.

PBMCs were activated for 2 days with medium containinglipopoly-saccharide (LPS), pokeweed mitogen (PWM) and phytohemagglutinin(PHA). Cell lines and activated PBMCs were harvested and mRNA or totalRNA was isolated. cDNA was generated from messenger or total RNA byusing the Invitrogen cDNA Cycle kit. By using primers specific for eachspecies, double-stranded cDNA was generated via PCR.

Example II Preparation and Screening of Round I NCSM Libraries

Nucleic acid libraries comprising recombinant nucleic acid sequenceswere generated by using the seven cloned cDNA wild-type CD80 nucleicacid sequences as parental sequences and applying recursive sequencerecombination methods as described above to such sequences. In oneaspect, libraries comprising chimeric nucleic acid sequences weregenerated by applying DNA shuffling procedures to the seven mammaliancDNA sequences as described previously in, e.g., Stemmer, W. (1994)Nature 370:389–391 (1994) and Crameri, A. et al. (1998) Nature391:288–91, each of which is incorporated herein by reference in itsentirety for all purposes. Sequencing of randomly selected chimericclones a recombinant library indicated that 12 out of 12 clonescomprised nucleotide fragments from at least two of the starting genes,illustrating efficient chimerism.

Initial screening of the resulting chimeric NCSM clones was based onbinding assays in which binding of a polypeptide encoded by a clonenucleic acid and expressed on the surface of a cell transfected for oneof the two B7-1 ligands, CD28 or CTLA-4, was evaluated. For a detailedreview of the binding assays, see the “Materials and Methods” sectionabove. In brief, cells from a human HEK 293 cell line were transfectedwith plasmid DNA of the resulting recombinant libraries of recursivelyrecombined NCSM polynucleotides. Library transfectants were incubatedwith soluble CD28-Ig and CTLA-4-Ig conjugated withfluorescence-indicators, and sorted according to their fluorescence viaFACS-sorting and 96-well format HTP transfections. The invention is notlimited by the choice of fluorescence indicator molecules used (i.e.,numerous indicator molecules may be used).

Flow cytometry-based cell sorting was used to screen the libraries forclones with increased or decreased relative binding to CD28 and CTLA-4.(Fluorescently-labeled soluble CD28-Ig and CTLA-4-Ig molecules were usedas soluble CD28 and CTLA-4 receptors, respectively, in the competitiveor individual binding assays.) Transfected cells that preferentiallybound CD28 over CTLA-4 (as compared to CD28 and CTLA-4 binding ofwild-type co-stimulatory B7-1 molecules, such as, e.g., hB7-1) in anindividual or competitive binding assay were sorted out from the librarytransfectants as were cells that preferentially bound CTLA-4 over CD28(again, as compared to CD28 and CTLA-4 binding of wild-type B7-1molecules, such as, e.g., hB7-1) in an individual or competitive bindingassay. A large fraction of the cell-surface displayed shuffled chimericmolecules displayed exhibited binding to either sCD28-Ig or sCTLA-4-1g.Less than 10% of the randomly selected chimeras demonstrated no bindingto either sCD28-Ig or sCTLA-4-Ig (data not shown), indicating highfunctional fitness of polypeptides and proteins generated by DNAshuffling of natural wild-type mammalian B7-1 genes.

Plasmid DNA encoding the recursively recombined NCSM molecules wasrecovered from both categories of sorted cells and DNA was prepared forbinding assays. In the binding assays, DNA from individual clones wastransfected into human HEK 293 cells (or COS or other cells ofinterest), and the cells were analyzed for the ability to preferentiallybind either fluorescent-labeled sCD28-Ig or fluorescent-labeledsCTLA-4-Ig (either separately or competitively), as compared to thebinding of cells expressing wild-type B7-1 to the labeled sCD28-Ig orsCTLA-4-Ig. Clones exhibiting preferential binding for either CD28 orCTLA, as compared to the binding of wild-type (WT) B7-1 to CD28 orCTLA-4, were then analyzed by DNA sequencing, amino acid sequencing, andfunctional assays as described below.

Subsequent analysis of 1000 individual clones recovered from the sortedcells identified a number of clones with altered ligand binding profilesfor CD28 and CTLA-4 relative to the binding of wild-type B7-1 to CD28and CTLA-4. Four clones with preferential binding to sCD28-Ig oversCTLA-4-Ig (compared to the relative binding of WT hB7-1 to sCD28-Ig andsCTLA-4-Ig) were subjected to more detailed analysis; reduced binding ofthese clones to sCTLA-4-Ig was observed in at least two separateexperiments, while their binding to sCD28-Ig remained intact (data notshown). Although the level of binding of these clones to sCTLA-4-Ig wasreduced, however, it was still detectable (data not shown). Preferentialbinding to sCTLA-4-Ig over sCD28-Ig (compared to the relative binding ofWT hB7-1 to sCD28-Ig and sCTLA-4-Ig) was observed in at least 15 clonesof the 1000 individual Round 1 clones analyzed by flow cytometry (datanot shown). Again, the loss of binding to sCD28-Ig was only partial.

Based on the results of this screening assay, exemplary NCSM moleculeshaving a desired phenotype comprising an altered CD28/CTLA-4 bindingaffinity ratio or CTLA-4/CD28 binding affinity ratio (relative to thatof WT hB7-1) were identified. Plasmid DNA encoding NCSM molecules ofthese selected clones was recovered and DNA was prepared for subsequentbinding and T cell proliferation functional assays.

In T cell proliferation assays, DNA from each of the selected exemplaryCD28BP or CTLA44BP clones identified in the binding assays wastransfected into either monkey (COS-7) or human embryonic kidney (human293 cell line) cells in vitro, using procedures described above, andtested for an ability to induce or inhibit T cell proliferation ascompared to human wild-type B7-1. Anti-CD28 mAbs can be used as apositive control. (An alternative functional test is to transfect orvaccinate human or animal cells in vivo with the nucleic acid from aselected clone, as described below.) To stimulate T cell activation viatwo-signaling pathway, human T cells were incubated with the cellsexpressing a selected NCSM molecule and anti-CD3 antibody. See “T CellProliferation Assays” in “Materials and Methods.” For example, byincubating T cells with anti-CD3 antibody and cells expressing a CD28BPof the invention, the cell surface-expressed CD28BP bound CD28 receptoron the T cells and the anti-CD3 bound the CD3 T cell receptor. This wasfollowed by addition of H³-thymidine to measure the increase in DNAsynthesis (a commonly used indication of cell proliferation), asdescribed previously. FIGS. 10 and 12 illustrate results of T cellproliferation assays.

Clones that induced T cell proliferation that was about equal to, equalto or higher than that induced by WT hB7-1 were identified and selectedfor further characterization from the recombinant nucleic acid librarypre-enriched by FACS sorting for clones having preferential binding toCD28 over CTLA-4. Four exemplary clones having the most biased bindingto CD28 and an ability to induce a cell proliferation response aboutequal to or higher than that induced by WT hB7-1 were identified anddesignated Round 1 (R1) CD28BP-71, -84, -118, and -126 clones. Aminoacid and nucleic acid sequences of these NCSM clones were determinedusing standard sequencing techniques described above.

Clones that reduced or suppressed T cell proliferation relative to thethat generated by WT human B7-1 were identified and selected for furthercharacterization from the recombinant nucleic acid library pre-enrichedby FACS sorting for clones having preferential binding to CTLA-4 overCD28. Five exemplary clones exhibiting the most biased binding to CTLA-4and the ability to reduce or suppress a T cell proliferation relative tothat generated by WT human B7-1 were identified and designated R1CTLA-4BP-5, -7, -11, -13, and -27 clones. The amino acid and nucleicacid sequences of these NCSM clones were determined using standardsequence techniques as described above.

Round 1 clones showed diverse amino acid and nucleotide differences fromwild-type B7-1 sequences. For example, R1 CTLA4BP clones demonstrated a93.4–97.6% amino acid identity with a wild-type B7-1 molecule and a97–98.5% identity at the nucleotide level. In competitive binding assaysusing CD28-Ig and CTLA-4-Ig, the R1 CTLA-4BPs displayed preferentialbinding to CTLA-4-Ig as compared to the wild-type B7-1 bindingpreference to CD28-Ig and CTLA-4-Ig.

Example III Preparation and Screening of Round 2 NCSM Libraries

The nucleotide sequences (or nucleotide segments or fragments thereof)corresponding to R1 CD28BP-71, -84, -118, and -126 clones were used asparental sequences for recursive sequence recombination in thegeneration of a second round CD28BP NCSM recombinant nucleic acidlibrary. The nucleotide sequences (or nucleotide segments or fragmentsthereof) corresponding to R1 CTLA-4BP-5, -7, -11, -13, and -27 cloneswere used as parental sequences for DNA shuffling in the generation of asecond round CTLA-4BP NCSM recombinant nucleic acid library. As in Round1, the nucleotide sequences corresponding to the selected CD28BP cloneswere recursively recombined with one another and the selected CTLA-4BPswere recursively recombined with one another by DNA shuffling inparallel experiments. Two separate sets of libraries were generated (onederived from the selected CD28BP parental clones and one derived fromthe selected CTLA-4BP clones). Binding assays were performed aspreviously described on the recombinant libraries generated in Round 2(e.g., libraries containing recursive sequence recombinants wereincubated with soluble CD28-Ig and CTLA-4-Ig conjugated withfluorescence indicators, and sorted according to their fluorescencebinding profiles).

Screening of 1000 individual clones from both libraries identified anumber of clones that exhibited strongly biased binding to eithersCD28-Ig or sCTLA-4-Ig. The second round of breeding resulted in anumber of different clones exhibiting biased (altered) binding tosCD28-Ig or sCTLA-4-Ig, respectively. For example, a number of Round 2(R2) CD28BP clones showed even greater preferential binding to CD28 thandid the R1 CD28BP clones. Similarly, a number of R2 CTLA-4BP clonesshowed even greater preferential binding to CTLA-4 than did the R1CTLA-4BP clones. From the shuffled recombinant libraries of the secondround of breeding, a number of clones with further biased binding tosCD28-Ig or sCTLA-4-Ig, respectively, as compared to the clones selectedfrom the first round of breeding (data not shown), were selected fromthe Round 2 libraries using the same criteria as in Round 1. The clonesdisplayed a range of expression on the stable transfectants as comparedto WT hB7-1 transfectants. The plasmid DNA for selected clones wasrecovered and DNA prepared for further binding and functional assays. Inaddition, the amino acid and nucleic acid sequences of these clones weredetermined using standard procedures known in the art.

A. Round 2 Clones With Preferential Binding Properties

Seventeen R2 CD28BP clones were found to have preferential binding toCD28 over CTLA-4 as shown in both individual and competitive bindingassays between these cells transfected with these clones and solubleCD28-Ig fusions and/or CTLA4-Ig fusions. Binding profiles for the 17clones are shown in FIG. 5. These clones were designated clones Round 2(R2) CD28BP-1 through CD28BP-17 and their respective amino acid andnucleic acid sequences were determined (see Table 3). Thirty-six R2CD28BP clones were also found to have preferential binding to CD28 overCTLA-4 relative to WT hB7-1 as demonstrated in both individual andcompetitive binding assays between these cells transfected with theseclones and soluble CD28-Ig fusions and/or CTLA4-Ig fusions. These cloneswere designated clones R2 CD28A12-5 through CD28E2-4 and theirrespective amino acid and nucleic acid sequences were determined (seeTable 3). Twelve R2 CD28BP clones binding profiles similar to that of WThB7-1 were also selected from both R2 libraries and their respectiveamino acid and nucleic acid sequences were determined. Binding profilesfor other recombinant CD28BP clones described herein were also generated(data not shown).

TABLE 3 Nucleic acid Protein Binding SEQ ID NO: SEQ ID NO: Clone IDScore SEQ ID NO: 1 SEQ ID NO: 48 R1clone71 1 SEQ ID NO: 2 SEQ ID NO: 49R1clone84 1 SEQ ID NO: 3 SEQ ID NO: 50 R1clone118 1 SEQ ID NO: 4 SEQ IDNO: 51 R1clone126 1 SEQ ID NO: 5 SEQ ID NO: 52 CD28BP1 2 SEQ ID NO: 6SEQ ID NO: 53 CD28BP2 2 SEQ ID NO: 7 SEQ ID NO: 54 CD28BP3 2 SEQ ID NO:8 SEQ ID NO: 55 CD28BP4 2 SEQ ID NO: 9 SEQ ID NO: 56 CD28BP5 2 SEQ IDNO: 10 SEQ ID NO: 57 CD28BP6 2 SEQ ID NO: 11 SEQ ID NO: 58 CD28BP7 2 SEQID NO: 12 SEQ ID NO: 59 CD28BP8 2 SEQ ID NO: 13 SEQ ID NO: 60 CD28BP9 2SEQ ID NO: 14 SEQ ID NO: 61 CD28BP10 2 SEQ ID NO: 15 SEQ ID NO: 62CD28BP11 2 SEQ ID NO: 16 SEQ ID NO: 63 CD28BP12 2 SEQ ID NO: 17 SEQ IDNO: 64 CD28BP13 2 SEQ ID NO: 18 SEQ ID NO: 65 CD28BP14 2 SEQ ID NO: 19SEQ ID NO: 66 CD28BP15 2 SEQ ID NO: 20 SEQ ID NO: 67 CD28BP16 2 SEQ IDNO: 21 SEQ ID NO: 68 CD28BP17 2 SEQ ID NO: 95 SEQ ID NO: 174 CD28A12-5 1SEQ ID NO: 96 SEQ ID NO: 175 CD28A4-5* 1 SEQ ID NO: 97 SEQ ID NO: 176CD28A4-9 1 SEQ ID NO: 98 SEQ ID NO: 177 CD28A6-9 1 SEQ ID NO: 99 SEQ IDNO: 178 CD28A6-1 1 SEQ ID NO: 100 SEQ ID NO: 179 CD28A8-4 1 SEQ ID NO:101 SEQ ID NO: 180 CD28A8-6 1 SEQ ID NO: 102 SEQ ID NO: 181 CD28B2-8 1SEQ ID NO: 103 SEQ ID NO: 182 CD28B4-3 1 SEQ ID NO: 104 SEQ ID NO: 183CD28B6-3 1 SEQ ID NO: 105 SEQ ID NO: 184 CD28B6-6 1 SEQ ID NO: 106 SEQID NO: 185 CD28B8-5* 1 SEQ ID NO: 107 SEQ ID NO: 186 CD28C11-5 2 SEQ IDNO: 108 SEQ ID NO: 187 CD28C6-1 1 SEQ ID NO: 109 SEQ ID NO: 188 CD28C7-31 SEQ ID NO: 110 SEQ ID NO: 189 CD28C8-6 2 SEQ ID NO: 111 SEQ ID NO: 190CD28C9-5* 1 SEQ ID NO: 112 SEQ ID NO: 191 CD28C2-4 1 SEQ ID NO: 113 SEQID NO: 192 CD28D2-3 1 SEQ ID NO: 114 SEQ ID NO: 193 CD28D2-9 1 SEQ IDNO: 115 SEQ ID NO: 194 CD28D8-9 1 SEQ ID NO: 116 SEQ ID NO: 195CD28D11-1 2 SEQ ID NO: 117 SEQ ID NO: 196 CD28D12-5 2 SEQ ID NO: 118 SEQID NO: 197 CD28E10-6 1 SEQ ID NO: 119 SEQ ID NO: 198 CD28F7-2 2 SEQ IDNO: 120 SEQ ID NO: 199 CD28F8-4 1 SEQ ID NO: 121 SEQ ID NO: 200CD28F10-2 1 SEQ ID NO: 122 SEQ ID NO: 201 CD28F12-5* 1 SEQ ID NO: 123SEQ ID NO: 202 CD28G2-8 1 SEQ ID NO: 124 SEQ ID NO: 203 CD28G1-5 1 SEQID NO: 125 SEQ ID NO: 204 CD28G1-9 1 SEQ ID NO: 126 SEQ ID NO: 205CD28H4-3 1 SEQ ID NO: 127 SEQ ID NO: 206 CD28H11-3 1 SEQ ID NO: 128 SEQID NO: 207 CD28H6-6 1 SEQ ID NO: 129 SEQ ID NO: 208 CD28E2-4 1 SEQ IDNO: 130 SEQ ID NO: 209 CD28B4-5a 1 SEQ ID NO: 131 SEQ ID NO: 210CD28A2-5 0 SEQ ID NO: 132 SEQ ID NO: 211 CD28B4-5* 0 SEQ ID NO: 133 SEQID NO: 212 CD28D5-6 0 SEQ ID NO: 134 SEQ ID NO: 213 CD28D10-4 0 SEQ IDNO: 135 SEQ ID NO: 214 CD28E2-5* 0 SEQ ID NO: 136 SEQ ID NO: 215CD28E5-2 0 SEQ ID NO: 137 SEQ ID NO: 216 CD28E8-6 0 SEQ ID NO: 138 SEQID NO: 217 CD28E9-6 0 SEQ ID NO: 139 SEQ ID NO: 218 CD28F3-1 0 SEQ IDNO: 140 SEQ ID NO: 219 CD28F3-5 0 SEQ ID NO: 141 SEQ ID NO: 220 CD28F3-60 SEQ ID NO: 142 SEQ ID NO: 221 CD28F11-8 0

Table 3 above presents a summary of the relative binding activities ofthese selected R2 CD28BP NCSM clones based on three exemplary bindingprofiles shown in FIGS. 6B(1)–6B(3). In the three exemplary bindingprofiles, the Y-axis represents binding to CD28, and the X-axisrepresents binding to CTLA-4. An exemplary binding profile for thebinding of WT B7-1 to CD28 and CTLA-4 is shown in FIG. 6B(1), indicatingapproximately equal binding affinity of WT B7-1 to CD28 and CTLA-4. Anexample of a binding profile indicating high preferential binding toCD28 over CTLA-4 relative to that of WT B7-1 is shown in FIG. 6B(3);that is, the clone has a CD28/CTLA-4 binding affinity ratiosignificantly greater than the CD28/CTLA-4 binding affinity ratio of WThB7-1. An example of a binding profile indicating intermediatepreferential binding to CD28 over CTLA-4 relative to that of WT B7-1 isshown in FIG. 6B(2) (i.e., a CD28/CTLA-4 binding affinity ratio greaterthan the CD28/CTLA-4 binding affinity ratio of WT hB7-1).

A score is assigned to each clone based upon comparison to the threeexemplary binding profiles. A score of zero (0) indicates the CD28BPclone has a binding profile equivalent or substantially equivalent tothat of WT B7-1. A score of 1 indicates the CD28BP clone has a bindingprofile similar to that shown in FIG. 6B(2) (i.e., CD28/CTLA-4 bindingaffinity ratio greater than the CD28/CTLA-4 binding affinity ratio of WThB7-1). A score of 2 indicates the clone has a binding profile similarto that shown in FIG. 6B(3) (i.e., a CD28/CTLA-4 binding affinity ratiosignificantly greater than the CD28/CTLA-4 binding affinity ratio of WThB7-1). Table 3 shows the clone identification (ID) name for eachselected R2 CD28BP clone and its score.

R2 CD28BP clones 3, 6, and 9 (corresponding to nucleic acid sequencesSEQ ID NOS:7, 10, and 13, and amino acid sequences 54, 57, and 60,respectively) comprise identical amino acid and nucleic acid sequences;the competitive binding assays and T cell proliferation assays for theseclones were conducted in a repeated manner to verify functionalactivity. Clones Round 2 CD28BP-1 and -12 comprise identical amino acidsequences (amino acid sequences SEQ ID NOS:52 and 63, respectively);however, the nucleic acid sequences of clones R2 CD28BP-1 and -12(nucleic acid sequences SEQ ID NOS:5 and 16, respectively) differ fromone another by one nucleic acid residue at position 894 in bothsequences. Clone 1 has nucleic acid residue C at position 894 (with theresulting codon TCC encoding Ser); clone 12 has nucleic acid residue Tat position 894 (with the resulting codon TCT also encoding Ser).

Fifty R2 CTLA-4BP clones were found to have preferential binding to CD28over CTLA-4 as shown in both individual and competitive binding assaysbetween cells transfected with these clones and fluorescently labeledsoluble CD28-Ig fusions and/or CTLA-4-Ig fusions. Exemplary bindingprofiles for selected clones are shown in FIGS. 7A–7H. The respectiveamino acid and nucleic acid sequences of the clones were determined(Table 4). Table 4 presents a summary of the relative binding activitiesof these selected 50 R2 CTLA-4BP clones based on the three exemplarybinding profiles shown in FIGS. 6A(1)–6A(3). In the three exemplarycompetitive binding profiles, the Y-axis represents binding to CD28, andthe X axis represents binding to CTLA-4 (see binding assays described in“Materials and Methods” ). An exemplary binding profile for the bindingof WT B7-1 to CD28 and CTLA-4 is shown in FIG. 6A(1), indicatingapproximately equal binding affinity of WT B7-1 to CD28 and CTLA-4. Anexemplary binding profile indicating for a particular clone apreferential binding to CTLA-4 over CD28relative to that of WT B7-1 isshown in FIG. 6A(3); the clone has a CTLA-4/CD28 binding affinity ratiosignificantly greater than the CTLA-4/CD28 binding affinity ratio of WThB7- 1. An exemplary binding profile indicating intermediatepreferential binding to CD28 over CTLA-4 relative to that of WT B7-1 isshown in FIG. 6A(2); the clone has a CTLA-4/CD28 binding affinity ratiogreater than that of WT hB7-1.

A score is assigned to each NCSM clone based upon comparison to thethree exemplary binding profiles. A score of zero (0) indicates theCTLA-4BP clone has a binding profile similar or equivalent to that of WTB7-1. A score of 1 indicates the CTLA-4BP clone has a binding profilesimilar or equivalent to that shown in FIG. 6A(2) (i.e., with aCTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CCD28 bindingaffinity ratio of WT hB7-1). A score of 2 indicates the CTLA-4BP clonehas a binding profile similar or equivalent to that shown in FIG. 6A(3)(i.e., with a CTLA-4/CD28 binding affinity ratio significantly greaterthan that of WT hB7-1). The clone identification (ID) name and scoreassigned to each selected CTLA-4BP clone are shown in Table 4. Bindingprofiles for other CTLA-4BP clones described herein were also generated(data not shown).

TABLE 4 Nucleic acid Protein Binding SEQ ID NO: SEQ ID NO: Clone IDScore SEQ ID NO: 22 SEQ ID NO: 69 R1-5 2 SEQ ID NO: 23 SEQ ID NO: 70R1-7 1 SEQ ID NO: 24 SEQ ID NO: 71 R1-11 1 SEQ ID NO: 25 SEQ ID NO: 72R1-13 1 SEQ ID NO: 26 SEQ ID NO: 73 R1-27 1 SEQ ID NO: 27 SEQ ID NO: 745x2-10c 2 SEQ ID NO: 28 SEQ ID NO: 75 5x2-11d 2 SEQ ID NO: 29 SEQ ID NO:76 5X2-12F 1 SEQ ID NO: 30 SEQ ID NO: 77 5x2-2g 2 SEQ ID NO: 31 SEQ IDNO: 78 5x2-3c 2 SEQ ID NO: 32 SEQ ID NO: 79 5x2-4c 1 SEQ ID NO: 33 SEQID NO: 80 5x2-7b 1 SEQ ID NO: 34 SEQ ID NO: 81 5x2-8c 2 SEQ ID NO: 35SEQ ID NO: 82 5x3-10e 2 SEQ ID NO: 36 SEQ ID NO: 83 5X3-11B 1 SEQ ID NO:37 SEQ ID NO: 84 5x3-6f 2 SEQ ID NO: 38 SEQ ID NO: 85 5X4-11D 2 SEQ IDNO: 39 SEQ ID NO: 86 5X4-12C 2 SEQ ID NO: 40 SEQ ID NO: 87 5x4-1F 2 SEQID NO: 41 SEQ ID NO: 88 5X5-2E 2 SEQ ID NO: 42 SEQ ID NO: 89 5X5-6E 2SEQ ID NO: 43 SEQ ID NO: 90 5X6-9D 2 SEQ ID NO: 44 SEQ ID NO: 91 5X8-1F2 SEQ ID NO: 45 SEQ ID NO: 92 5X9-12C 2 SEQ ID NO: 143 SEQ ID NO: 2225x9-d10 1 SEQ ID NO: 144 SEQ ID NO: 223 5x6-f6 1 SEQ ID NO: 145 SEQ IDNO: 224 5x5-h12 1 SEQ ID NO: 146 SEQ ID NO: 225 5x5-c10 1 SEQ ID NO: 147SEQ ID NO: 226 5x3-e8 1 SEQ ID NO: 148 SEQ ID NO: 227 5x3-c4 1 SEQ IDNO: 149 SEQ ID NO: 228 5x3-c3 1 SEQ ID NO: 150 SEQ ID NO: 229 5x2-h11 1SEQ ID NO: 151 SEQ ID NO: 230 5x2-d7 1 SEQ ID NO: 152 SEQ ID NO: 2315x2-b7 1 SEQ ID NO: 153 SEQ ID NO: 232 5x2-b1 1 SEQ ID NO: 154 SEQ IDNO: 233 5x1-f1 1 SEQ ID NO: 155 SEQ ID NO: 234 5x1-d7 1 SEQ ID NO: 156SEQ ID NO: 235 2x4-g9 1 SEQ ID NO: 157 SEQ ID NO: 236 2x4-a6 1 SEQ IDNO: 158 SEQ ID NO: 237 2x2-f3 1 SEQ ID NO: 159 SEQ ID NO: 238 2x2-f12 1SEQ ID NO: 160 SEQ ID NO: 239 2x1-g8 1 SEQ ID NO: 161 SEQ ID NO: 2402x1-f10 1 SEQ ID NO: 162 SEQ ID NO: 241 2x1-c9 1 SEQ ID NO: 163 SEQ IDNO: 242 2x1-h12 1 SEQ ID NO: 164 SEQ ID NO: 243 2x1-e2 1 SEQ ID NO: 165SEQ ID NO: 244 2x1-c4 1 SEQ ID NO: 166 SEQ ID NO: 245 2x1-b12 1 SEQ IDNO: 167 SEQ ID NO: 246 2x2-f1 1 SEQ ID NO: 168 SEQ ID NO: 247 5X4-h1 2SEQ ID NO: 169 SEQ ID NO: 248 5x4-a1 0 SEQ ID NO: 170 SEQ ID NO: 2495x2-f3 0 SEQ ID NO: 171 SEQ ID NO: 250 5x2-e12 0 SEQ ID NO: 172 SEQ IDNO: 251 2x4-h11 0 SEQ ID NO: 173 SEQ ID NO: 252 2x3-h2 0 SEQ ID NO: 253SEQ ID NO: 263 A-H3-6 1 SEQ ID NO: 254 SEQ ID NO: 264 A-B11-5 1 SEQ IDNO: 255 SEQ ID NO: 265 A-E2-6 1 SEQ ID NO: 256 SEQ ID NO: 266 A-F1-6 1SEQ ID NO: 257 SEQ ID NO: 267 A-F6-9 1 SEQ ID NO: 258 SEQ ID NO: 268A-H4-5* 1 SEQ ID NO: 259 SEQ ID NO: 269 A-B4-6 1 SEQ ID NO: 260 SEQ IDNO: 270 A-F10-1 1 SEQ ID NO: 261 SEQ ID NO: 271 A-G8-1 1 SEQ ID NO: 262SEQ ID NO: 272 A-C9-9 0

The plasmids corresponding to the 36 clones displaying preferentialbinding to CD28 over CTLA-4 relative to that of WT hB7-1 were recovered;each corresponding CD28BP molecule was recovered, and nucleic acid andamino acid sequences were determined. The clones were assigned scores of2 and 1, as shown in Table 3, depending upon the magnitude of theobserved preferential binding.

The plasmids for the 12 R2 clones from the CD28BP R2 library thatdisplayed CD28 and CTLA-4 binding profiles equal or approximatelyequivalent to that of WT B7-1 were recovered; each corresponding clonemolecule was recovered and its nucleic acid and amino acid sequenceswere determined. These clones were assigned a score of zero, as shown inTable 3.

Two groups of R2 CTLA-4BP clones (19 in the first group, Group I, and 26in the second group, Group II) were identified as having preferentialbinding to CTLA-4 over CD28 relative to that of hB7-1 as demonstrated incompetitive binding assays between cells transfected with these clonesand fluorescently labeled soluble CD28-Ig fusions and/or CTLA4-Igfusions. These clones are identified in Table 4, which indicates theclone ID name and binding score (as explained with regard to Table 3);the amino acid and nucleic acid sequences for each CTLA-4BP clone weredetermined. In general, the R2 clones displayed a greater degree ofCTLA-4 binding preference than the R1 clones. In addition, five R2clones from the CTLA-4 R2 library with binding profiles equal orapproximately equivalent to that of WT human B7-1 were identified.Plasmids for all such clones were recovered; the nucleic acid and aminoacid sequences were determined for each clone, and each clone wasassigned a binding score (see Table 4).

Preferred or enhanced binding of a selected clone to CD28 over CTLA-4relative to hB7-1 was observed by an increase in the observedCD28/CTLA-4 binding affinity ratio for the clone compared to theCD28/CTLA-4 binding affinity ratio of hB7-1. Preferred or enhancedbinding of a selected clone to CTLA-4 over CD28 relative to hB7-1 isobserved by an increase in the observed CTLA-4/CD28 binding affinityratio for the clone compared to the CTLA-4/CD28 binding affinity ratioof hB7-1.

The R2 selected clone that exhibited the greatest increase inCD28/CTLA-4 binding affinity ratio compared to the CD28/CTLA-4 bindingaffinity ratio of hB7-1 was clone R2 CD28BP-15. The identified clonethat exhibited the greatest increase in CTLA-4/CD28 binding affinityratio compared to the CTLA-4/CD28 binding affinity ratio of hB7-1 wasclone R2 CTLA-4BP 5x4-12c. FIGS. 4A–4D show a characterization ofcompetitive ligand binding properties of CD28BP-15 and CTLA-4BP 5x4-12ccompared to human B7-1. 293 cells transfected with CD28BP-15, CTLA-4BP5x4-12c, hB7-1, or a negative control vector (which did not contain theB7-1, CTLA4-BP, or CD28BP nucleotide sequence insert) were stained withfluorescently labeled biotin-conjugated sCD28-Ig or FITC-conjugatedCTLA-4-Ig fusion proteins. The results of a representative flowcytometry analysis are shown in FIGS. 4A–4D; similar results wereobtained in five other experiments. Transfectants expressing CD28BP-15or CTLA-4BP 5x4-12c, and stained under identical conditions,demonstrated dramatically altered ligand binding profiles, as comparedto transfectants expressing hB7-1 stained in an identical manner. Inparticular, CD28BP-15 showed a significantly changed binding profile toCD28 as compared to the binding of hB7-1 to CD28. CD28BP-15 also showeda significant loss of CTLA-4 binding as compared to the binding of humanB7-1 to CTLA-4.

FIGS. 8A–8B show the respective amino acid sequences for CD28BP-15 andCTLA-4BP 5x4-12c and the genealogy of these sequences. The nucleotideand amino acid sequences for each of CD28BP-15 and CTLA-4BP 5x4-12c werealigned with the starting genes to identify the parental origins of therecombinant sequences. The chimeric nature of each recombinant aminoacid sequence is indicated by a solid labeled line designating eachamino acid subsequence derived from a particular parental speciessequence. Any amino acid residue that differs from a residue in the WThuman B7-1 sequence in the corresponding (equivalent) amino acid residueposition is indicated with a star (*). Three point mutations in CTLA-4BP5x4-12c that were not derived from any of the starting parental genesare indicated with a solid triangle. The predicted transmembrane domainis illustrated with a dashed line (prediction based on equivalentanalysis for mammalian B7-1 molecules in Parsons, K. R. & Howard, C. J.(1999) Immunogenetics 49:231–4).

Sequence analysis of CD28BP-15 indicated the amino acid sequencecomprised a chimera derived principally from human, bovine, and rabbitsequences (FIG. 8B). The remaining clones selected from the librariesresulting from second round of recombination that displayed preferentialbinding to CD28 over CTLA-4 shared about 74 to about 99% sequenceidentity with CD28BP-15 based on sequence alignment comparisons (usingDNASTAR or Vector NTI algorithm with default parameters as describedabove), illustrating the diversity of clones having the preferentialbinding properties. The nucleotide and amino acid sequence identities ofCD28BP-15 with human B7-1 were 73% and 61%, respectively (using DNASTARor Vector NTI algorithm with default parameters). Based on amino acidsequence alignments, all of the selected CD28BP clones exhibitingpreferential binding to CD28 over CTLA-4 relative to hB7-1 contained asubstitution of valine for isoleucine at amino acid position 49(Ile49Val) of the mature CD28BP-15 amino acid sequence (corresponding toalignment with the mature hB7-1 sequence). This substitution correspondsto a substitution at amino acid position 85 in the full-length CD28BP-15and amino acid position 83 in full-length human B7-1, since CD28BP-15includes two additional amino acid residues in the putative sequence.Although Ile49 does not appear to be directly involved in theinteraction of B7-1 with CTLA-4 (see, e.g., Stamper, C. et al. (2001)Nature 410:608–611), substitution Ile49Ala was previously shown tocompletely abolish binding of B7-1 to both CD28 and CTLA-4, suggestingthe importance of this residue in the ligand binding (Peach, R. J. etal. (1995) J Biol Chem 270:21181–7). Although the Ile49Val substitutionis also considered a conservative replacement, our data suggest thatmutations derived from naturally existing genes, in this case from thebovine B7-1 gene sequence, provide improved means to search for alteredfunctional properties in proteins. Notably, bovine CD28 receptor is theonly exception among CD28 and CTLA-4 receptor amino acid sequencesanalyzed from 12 different species in which the hexapeptide MYPPPY (SEQID NO:297) and Gly-66 are not fully conserved in the receptor sequence(i.e., MYPPPY is replaced by LYPPPY (SEQ ID NO:312), and Gly-66 isreplaced by valine (see Metzler, W. J. et al. (1997) Nat Struct Biol4:527–31). The hexapeptide MYPPPY is conserved in the F-G loop of bothCD28 and CTLA-4 in a variety of mammalian species. Mutation of anyresidue in the MYPPPY sequence leads to reduced binding to B7-1 and B7-2(see id.), and all these residues, with the exception of Pro101, are indirect contact with B7-1 (Stamper, C. et al. (2001) Nature 410:608–611),suggesting that changes in this region of bovine CD28 receptor havedriven the natural evolution of bovine B7-1 to acquire properties thatalso benefited the in vitro evolution of CD28BP described herein.

CTLA-4BP 5x4-12c contained amino acid sequences correspondingprincipally to the human, baboon, rhesus monkey and bovine B7-1 genes(FIG. 8A), and the nucleotide and amino acid sequences of CTLA-4BP were97% and 96% identical with those of hB7-1, respectively. In addition,CTLA-4BP 5x4-12c contained three amino acid mutations that were notderived from any of the starting genes (FIG. 8A). The remaining cloneswith preferential binding to sCTLA-4-Ig over sCD28-Ig in the bindingassays exhibited 95–99% identity with CTLA-4BP 5x4-12c (e.g., usingDNASTAR or Vector NTI algorithm with default parameters).

Tyr-31 in the mature sequence of CTLA-4BP 5x4-12c (as measured from theN-terminus of the mature domain, using the amino acid position andnumbering corresponding to the amino acid sequence of hB7-1 (SEQ IDNO:278) was replaced by histidine (i.e., Tyr31His); the Tyr31Hissubstitution was also present in a number of selected recombinant cloneswith preferential binding to CTLA-4, whereas all selected clones withpreferential binding to CD28 retained the tyrosine at that position.Tyr-31 was also present at an equivalent position in all of the matureparental sequences. Tyr-31 of the mature CTLA-4BP and mature hB7-1sequences (as measured from the N-terminus of the mature domain, usingthe amino acid position and numbering corresponding to the amino acidsequence of hB7-1) corresponds to Tyr-65 of the full-length CTLA4-BP andhB7-1 sequences, respectively (as measured from the N-terminus, whichincludes the signal peptide sequence). DNA sequence analysis of CTLA-4BP5x4-12c showed a mutation in the codon corresponding to amino acidposition 31 as measured from the N-terminus in mature hB7-1—TAC—to codonCAC (at the corresponding position in mature form of CTLA-4BP 5x4-12c).Interestingly, it has been suggested that Tyr31 in human B7-2 may bereplaced by phenylalanine without any apparent change in the ligandbinding affinities (see, e.g., Freeman, G. J. et al. (1993) Science 262:909–11); Azuma, M. et al. (1993) Nature 366:76–9), whereas a Tyr3Alasubstitution in hB7-1 appears to completely abolish the binding of hB7-1to both CD28 and CTLA-4 (see Peach, R. J. et al. (1995) J Biol Chem270:21181–7). The present data demonstrate that the Tyr31Hissubstitution, at least when present in the context of the shuffledCTLA-4BP sequence, does not significantly affect interaction withCTLA-4, whereas this mutation appears to contribute to the loss inbinding to CD28, further supporting the suggestion that this residueplays an important role in the ligand binding of B7-1. Informationregarding the three-dimensional structures of CD28BP and CTLA-4BP isuseful in characterizing further the amino acid residues and structuresthat contribute to the preferential binding of NCSMs to their tworespective ligands.

Sequence Round 2 CD28BP clones showed a range of amino acid andnucleotide diversity from wild-type B7-1 molecules. For example, theRound 2 CTLA4BP clones had a 93.8–96.9% amino acid identity with a WTB7-1 molecule and had a 96.2–97.7% identity at the nucleotide level.

The amino acid sequence of clone R2 CD28BP-15 differs from that ofidentical clones R2 CD28BP-3, -6, and -9 by only one amino acid residueat position 110; CD29BP-15 has a proline residue at position 110; clonesCD28BP-3, -6, and -9 include a leucine residue at position 110.

The binding of labeled soluble ligand sCD28-Ig and sCTLA-4-Ig to clonesCD28BP-15 and CTLA-4BP 5x4-12c was further studied, as shown in FIGS.9A–9H. Specifically, 293 cells were transiently (FIGS. 9A–9B) or stably(FIGS. 9C–9D) transfected with CD28BP-15 (solid circles) or hB7-1 (opensquares), and with (dashed lines) and without (solid lines) a FLAG™ tag.293 cells were stably transfected with CTLA-4BP 5x4-12c (solidtriangles) or WT hB7-1 (open squares) (FIGS. 9D–9E). Cells transiencly(FIGS. 9A–9B) or stably (FIGS. 9C, 9D, 9E, 9F) transfected with a vectorlacking a WT hB7-1, CD28BP or CTLA-4BP nucleic acid insert were used asnegative controls (open diamonds). The transfectants were stained withincreasing concentrations of labeled soluble CD28-Ig (FIGS. 9A, 9C, 9E)or soluble CTLA4-Ig (FIGS. 9B, 9D, 9F), prepared as described above, andthe cells were analyzed by flow cytometry. Stable 293 transfectantsexpressing CTLA-4BP 5x4-12c (gray histograms), hB7-1 (gray histograms)and negative control transfectants (open histograms) were stained withanti-hB7-1 mAbs (FIGS. 9G–9H), and the expression levels were analyzedby flow cytometry.

Staining of transfectants individually with sCD28-Ig or sCTLA-4-Igindicated that CD28BP-15 transfectants bound sCD28-Ig at higher levelsthan transfectants expressing hB7-1 (as shown by increased MFI values),while sCTLA-4-Ig binding was significantly reduced, yet detectable(FIGS. 9A–9D) (as indicated by reduced MFI). On the other hand, littleor virtually no binding of sCD28-Ig to CTLA-4BP 5x4-12c transfectantswas detected even at high sCD28-Ig concentrations, while the sametransfectants did bind sCTLA-4-Ig, although at somewhat lower levelsthan the transfectants expressing hB7-1 (FIGS. 9E–9F) (as shown by MFIvalues). The lack of binding of sCD28-Ig to CTLA-4BP 5x4-12c was not dueto the lack of expression, as was determined by the binding ofanti-hB7-1 monoclonal antibody (mAb) to the transfectants (FIGS. 9G–9H).In contrast to CTLA-4BP 5x4-12c, mAbs specific for hB7-1 did notrecognize CD28BP (data not shown).

To analyze whether increased expression of CD28BP-15 on the transfectedcells contributed to the improved binding of sCD28-Ig, we fused aFLAG-tag (see according to Brizzard, B. L. et al. (1994) Biotechniques16:730–5) to hB7-1 and the shuffled clones, and the binding of aFLAG-specific mAb M2 (see id.) to the corresponding transfectants wasstudied. In seven separate transient transfections, the meanfluorescence intensity (MFI) of 293 cells transfected with CD28BP-FLAGand hB7-1-FLAG was 52±20 and 55±12, respectively (mean±SEM). Although nosignificant difference in the expression of the FLAG-constructs wasobserved, significantly improved binding of CD28-Ig was also observedusing the FLAG-constructs (FIGS. 9A–9B), strongly suggesting theimproved binding of sCD28-Ig by CD28BP-15 was due to improved affinityrather than improved expression of CD28BP-15.

We also analyzed the ligand binding properties of all starting B7-1genes by two-color analysis using sCD28-Ig and sCTLA-4-Ig to illustratethat CD28BP-14 and CTLA-4BP 5x4-12c showed evidence of truly newproperties. 293 cells transfected with human, rhesus monkey, orangutan,and baboon B7-1 genes exhibited essentially identical binding profilesto SCD28-Ig and sCTLA-4-Ig, whereas feline B7-1 showed little or nobinding to sCD28-Ig or sCTLA-4-Ig (data not shown). Bovine and rabbitB7-1 transfectants demonstrated sCD28-Ig binding in the same range asthat observed for human and primate B7-1, but their level of binding tosCTLA-4-Ig was somewhat lower. However, the binding level of sCTLA-4-Igto CD28BP-15 was consistently less than that of any of the otherstarting genes; the mean MFIs of sCTLA-4-Ig binding to human (n=4),bovine (n=4), rabbit B7-1 (n=2), and CD28BP-15 (n=2) were 247, 102, 205,and 30, respectively. Thus, CD28BP-15 and CTLA-4BP 5x4-12c displayedproperties that were unique as compared to any of the starting genes.

B. Functional Assays.

To investigate the functional properties of shuffled R2 CD28BPmolecules, nucleic acid sequences corresponding to representative clonesR2 CD28BP-1 to R2 CD28BP-17 were transfected into B7-negative cell linesand the capacity of the resulting transfectants to activate human Tcells was analyzed (using T cell proliferation assays with ³H thymidineincorporation as described above). Representative results of selectedclones are shown in FIG. 10. When used to costimulate T cells in solubleanti-CD3 induced proliferation assays, these selected clones induced arange of proliferative responses from slight induction (potentialantagonist) to improved induction over human B7-1 (FIG. 10). At one endof the range, clone CD28BP-8 (amino acid SEQ ID NO:59), which was shownto bind CD28, induced low T cell proliferation as compared to thewild-type hB7-1. CD28BP-8 induced a T cell proliferation level slightlygreater than that induced by cells transfected with an empty vector(lacking a B7-1, CD28BP, or CTLA-4BP nucleic acid insert). In contrast,CD28BP-15 induced a T cell proliferation response significantly greaterthan that induced by WT hB7-1 (FIG. 10). Other CD28BP clones induced a Tcell proliferation response about equal to that induced by WT hB7-1.

The enhanced co-stimulation of purified human T cells by clone CD28BP-15was further investigated as follows (FIGS. 11A–11C). 293 cells weretransiently (FIG. 11A) or stably (FIG. 11B) transfected with CD28BP-15nucleic acid, hB7-1 nucleic acid, or an empty control vector lacking theB7-1 or CD28BP-15 nucleic acid insert, and the irradiated transfectantswere co-cultured with purified human T cells and anti-CD3 mAbs (toinduce costimulation of T cells via the TCR) as described above.Mean±SEM (standard error of mean) of counts per minute (C.P.M.) (³Hthymidine incorporation) obtained in three (FIG. 11A) or six (FIG. 11B)independent experiments are shown.

CD28BP-15 transfectants induced greatly increased proliferation ofpurified human T cells cultured in the presence of anti-CD3 mAbscompared to cells transfected with hB7-1 (FIG. 11A). Moreover, wegenerated stable transfectants of CD28BP-15 and hB7-1 by selectingclones that expressed similar levels of the NCSM molecules based onbinding of sCD28-Ig at saturating concentrations. Similar to transienttransfectants, stable transfectants expressing CD28BP-15 induced a morepotent T cell proliferation than those transfectants expressing hB7-1(FIG. 11B).

Irradiated stable transfectants expressing CD28BP-15 or hB7-1 andnegative control cells transfected with a vector lacking the insert wereco-cultured with purified human T cells, and the levels of IFN-gammaproduction were measured after a culture period of 48 hours. Arepresentative experiment is shown in FIG. 11C; similar data wereobtained in three other experiments. Production of IFN-gamma in responseto transfectants expressing CD28BP-15 was higher than that induced byhB7-1 transfectants (FIG. 11C). Approximately 10-fold fewer transient orstable transfectants expressing CD28BP-15 than those expressing hB7-1were required to obtain a similar level of human T cell proliferation.Importantly, the maximum levels of T cell proliferation and IFN-gammaproduction were also increased (FIG. 11C). We believe these results arelikely attributable to the lack of negative signaling through CTLA-4.The increased affinity to CD28 and reduced affinity to CTLA-4 appears tohave contributed to the CD28BP-15-mediated improved T cell response.

The effect of Round 2 CTLA-4BP clones on purified T cells in T cellproliferation assays was investigated. In a representative T cellproliferation study performed as described above, 19 selected clones ofthe R2 library (designated as CTLA4BP-1 through CTLA4BP-19) havingreduced binding to CD28, but relatively strong binding to CTLA-4, werefound to reduce T cell proliferation in a soluble anti-CD3 induced Tcell proliferation assay (see FIG. 12) compared to that induced bywild-type hB7-1. Cells transfected with an empty vector (lacking a B7-1or CTLA-4BP nucleic acid insert), were used as the control. Theseselected CTLA-4BP clones produced a range of responses from reduced orsuppressed induction of T cells relative to hB7-1 induction, to asignificant inhibition of T cell proliferation (FIG. 12).

The effect of exemplary clone CTLA-4BP 5x4-12c on purified T cells andon T cell proliferation and cytokine synthesis induced in MLR wasfurther investigated. Representative results are shown in FIGS. 13A–13D.In the co-stimulation experiments, purified T cells were co-cultured inthe presence of soluble anti-CD3 mAbs (5 micrograms/ml) and transienttransfectants expressing hB7-1, CD28BP-15, CTLA-4BP 5x4-12c, or a vectorcontrol lacking the expressed sequence (FIG. 13A); a representativeexperiment is shown. Similar data were obtained in two otherexperiments. Increasing numbers of 293 cells stably transfected withCTLA-4BP 5x4-12c nucleic acid (solid triangles), hB7-1 nucleic acid(open squares), or a control vector lacking a B7-1, CTLA-4BP, or CD28BPnucleic acid insert (open diamonds) were added to the MLR cultures, asshown in FIG. 13B. The data represent mean±SEM of C.P.M. obtained in sixseparate MLR cultures, each performed using 4–6 replicate wells. MLR wascultured in the presence of 25000 irradiated 293 cells stablytransfected with hB7-1, CTLA-4BP 5x4-12c or a control vector without aninsert, as shown in FIGS. 13C–13D. IFN-gamma and IL-10 levels weremeasured by ELISA after an MLR culture period of 48 hours (FIGS.13C–13D). Six independent experiments were performed and the valuesobtained within one experiment are connected with a solid line. Theproduction levels of IFN-gamma significantly increased (P<0.05) andthose of IL-10 significantly decreased (P<0.01) for CTLA-4BP 5x4-12ccompared to hB7-1 or vector control (paired Student's t-test).

Relative to CD28BP-15, hB7-1, and a control vector, CTLA-4BP 5x4-12cexhibited very little ability to co-stimulate human T cells cultured inthe presence of soluble anti-CD3 mAbs (FIG. 13A). No co-stimulation ofhuman T cells was induced by either transient or stable CTLA-4BP 5x4-12ctransfectants, although efficient expression of the molecule on thesurface of the transfectants was observed using anti-B7-1 mAbs (seeFIGS. 9G–9H, FIG. 13A, and data not shown). In fact, only 2 of 19selected Round 2 clones (from Group I of the second round ofrecombination) that displayed preferential binding to CTLA-4 over CD28had the capacity to induce a T cell response that was more than 10% ofthat induced by hB7-1 (data not shown), illustrating the lack ofsCD28-Ig binding to these clones correlated with the lack of signalingthrough CD28.

More importantly, CTLA-4BP 5x4-12c transfectants inhibited T cellproliferation induced in a mixed lymphocyte reaction (MLR) in adose-dependent manner (FIG. 13B). Moreover, CTLA-4BP 5x4-12ctransfectants induced IL-10 production in MLR, but reduced IFN-gproduction, compared to hB7-1 or control transfectants (FIGS. 13D and13C, respectively), further supporting the notion that CTLA-4BP 5x4-12chas a dramatically altered biological function as compared to hB7-1.Several studies have suggested supporting roles for CTLA-4 (see, e.g.,McAdam, A. J. et al. (1998) Immunol Rev 165:231–47; Waterhouse, P. etal. (1995) Science 270:985–8; Perez, V. L. et al. (1997) Immunity6:411–7; Shrikant, P. et al. (1999) Immunity 11:483–93; van Elsas, A. etal. (1999) J Exp Med 190:355–66; Greenwald, R. et al. (2001) Immunity14:145–155) and IL-10 (see, e.g., Groux, H. et al. (1997) Nature389:737–42; Rizzo, L. V. et al. (1999) J Immunol 162:2613–22) ininducing and maintaining immunological tolerance. The results of thepresent invention also suggest that CTLA-4BPs of the invention,including, e.g., CTLA-4BP 5x4-12c, are useful in downregulating thefunction of specific T cells in autoimmune diseases and the like, andare thus of benefit in therapeutic and prophylactic methods for treatingsuch diseases.

Previous studies on B7 mutants showed that binding sites for CD28 andCTLA-4 are largely overlapping and that mutations in B7 that affectedthe binding to one ligand (i.e., CD28 or CTLA-4) generally also affectedthe binding to the other ligand. However, only a limited number ofvariants were tested and they were generally designed based oninformation of expected ligand binding sites. For example, in contrastto the present results, mutations of human B7-1 (hB7-1) that werepreviously designed based on structural information and predictedreceptor binding sites generally produced equivalent effects on bindingto CD28 and CTLA-4 (see, e.g., Peach, R. J. et al. J Biol Chem 270,21181–7 (1995). The current data show that the frequency of functionalvariants with altered ligand binding properties is sufficiently highenough that a desired phenotype may be rapidly identified using theappropriate selection criteria and screening procedures. Thus,appropriate screening of the sequences produced by recombination ofknown mammalian genomes serves as an effective means to identify thoserecombined sequences having novel functional properties without detailedknowledge of the receptor binding sites or the structures of theproteins. These results illustrate the advantages of the crossbreedingof mammalian genes of different species to evolve proteins having novelfunctional properties. CD28BP and CTLA-4BP may also help in furtherdeducing the mechanisms by which CD28 and CTLA-4 trigger positive andnegative signals to T cells, respectively. The fact that B7-1 and B7-2naturally bind to both CD28 and CTLA-4 has limited the potential scopeof their clinical applications. The chimeric CD28BP and CTLA-4BPmolecules described herein have wide-ranging clinical applications; theyare useful, for example, in the manipulation of T cell responses invitro and in vivo in a variety of pharmaceutical and medicalapplications and vaccinations.

Example IV Production of Soluble NCSM Polypeptides and Fusion Proteinsand Nucleic Acids Encoding Them

The present invention also provides soluble NCSM polypeptides, andsubsequences and fragments thereof that encompass a variety of formats,described herein, including, but not limited to, e.g., at least one ofan extracellular domain (ECD), of a NCSM polypeptide or a fragment,variant or homologue thereof, and an NCSM -ECD-Ig fusion protein orfragment, variant, or homologue thereof; and nucleotide andpolynucleotide sequences encoding all such polypeptides, proteins,fragments, homologues, and variants.

A soluble form of an NCSM polypeptide is useful in in vivo, ex vivo, orin vitro methods, including, e.g., therapeutic or prophylactic treatmentor diagnostic methods, administered as either a DNA plasmid expressionvector (e.g., DNA vaccine; gene therapy applications) or protein, fortreating or preventing various immunological disorders and diseases,including, e.g., cancers, including, but not limited to, e.g.,colorectal cancer, breast cancer, pancreatic cancer, lung cancer,prostate cancer, naso-pharyngeal cancer, cancer, brain cancer, leukemia,melanoma, head- and neck cancer, stomach cancer, cervical cancer,ovarian cancer, lymphomas, colon cancer, colorectal); allergy/asthma,autoimmune diseases, organ transplantation (e.g., graft versus hostdisease, and autoimmune diseases), chronic infectious diseases,including, but not limited to, e.g., viral infectious diseases, such asthose associated with, but not limited to, e.g., hepatitis B virus(HBV), herpes simplex virus (HSV), hepatitis C virus (HCV), HIV, humanpapilloma virus (HPV), and the like, and bacterial infectious diseases,such as, but not limited to, e.g., Lyme disease, tuberculosis, andchlamydia infections; and other diseases described herein fornon-soluble NCSMs of the invention, and as vaccine adjuvants in vaccineapplications as described for non-soluble NCSMS. Soluble NCSMs are alsouseful for diagnostic purposes, as for in vitro applications for testingand diagnosing such diseases.

In this example, selected CD28BP and CTLA-4BP clones were used for thepreparation of corresponding soluble CD28BP and CTLA-4BP polypeptidesand nucleic acids encoding such polypeptides. A CD28BP ECD polypeptideor fragment thereof may have a CD28/CTLA-4 binding affinity ratio aboutequal to, equal to or greater than the CD28/CTLA-4 binding affinityratio of hB7-1, and other biological properties described below; aCTLA-4BP ECD polypeptide of fragment thereof may have a CD28/CTLA-4binding affinity ratio equal to or greater than that of hB7-1, and otherbiological properties described below.

A. Expression Vectors Encoding Soluble ECDs of NCSM Polypeptides

Mammalian expression plasmids encoding soluble (non-membrane bound)extracellular domains (sECDs) of NCSM polypeptides (or fragmentsthereof) were constructed by PCR amplification using pfu turbopolymerase (Stratagene, La Jolla, Calif.) of selected NCSM ECDs fromplasmids containing a selected full-length NCSM DNA sequence. The PCRprimers were designed to specifically anneal with the first or last20–24 nucleotides of a particular nucleic acid region corresponding to aNCSM ECD (or fragment thereof), and flanked by restriction sites NheIand NotI, at their 5′ and 3′ ends, respectively. Amplicons of ˜730 basepairs (bp) (specific base pair numbers for each clone are shown in Table5) encoding individual ECDs (or fragments thereof) were digested withNheI and NotI (New England BioLabs, Beverly, Mass.), subsequentlypurified by low melt agarose gel electrophoresis (see proceduredescribed in Sambrook, supra). A pcDNA3.1(+) expression plasmid(Invitrogen, Carlsbad, Calif.), which was used as the backbone vector,was digested with NotI and ApaI restriction enzymes and fused (ligated)in-frame to a nucleic acid sequence encoding a carboxy-terminal epitopefusion tag comprised of the E-epitope (Amersham Pharmacia Biotech) andhexa-His tag using standard cloning procedures. This vector fragment wasthen digested with NheI and NotI and to an ˜5400 bp NheI-NotI fragmentcomprising the vector backbone and E-epitope/His-tag fusion sequence.This vector fragment was gel purified and ligated to each ˜730 bpNheI-NotI NCSM ECD polynucleotide to produce soluble ECD NCSM expressionplasmids. Mammalian expression plasmids encoding a soluble ECD form ofwild-type human B7-1 were similarly prepared from plasmids containingthe full-length WT human B7-1 DNA sequence.

Table 5

Nucleotide Amino Acid Clone ID Position Position 3′ end ECD wild-typehuB7.1-ECD 1-726 1-242 PDN CD28BP-8 ECD 1-735 1-245 IDQ CD28BP-11 ECD1-732 1-244 IDQ CD28BP-15 ECD 1-735 1-245 IDQ CTLA4-5X2-8C ECD 1-7261-242 PDN CTLA4-5X2-10C ECD 1-726 1-242 PDN CTLA4-5X4-1F ECD 1-726 1-242PDN CTLA4-5X4-11D ECD 1-726 1-242 PDN CTLA4-5X4-12C ECD 1-726 1-242 PDNCTLA4-5X5-2E ECD 1-726 1-242 PDN CTLA4-5X5-6E ECD 1-726 1-242 PDNCTLA4-5X6-9D ECD 1-726 1-242 PDN CTLA4-5X8-1F ECD 1-726 1-242 PDNNucleotide Amino Acid Clone ID Position Position 5′ end Fc P01857 HuIgG1-Fc 298–690 100–230 PKSCDKTH . . .

Table 5 shows for positions of nucleotide residues and correspondingamino acid residues of the signal peptide sequence and representativeECD domains of selected CD28BP and CTLA-4BP clones, and equivalentpositions in WT hB7-1 ECD, and the last three amino acid residues at the3′ end of each ECD of a selected NCSM clone or WT hB7-1. The presentinvention provides for ECD domains of the NCSM polypeptides (and nucleicacid sequences encoding such polypeptides) that lack the signal peptidesequence, such that the first about 33 or about 34 amino acids (or about99 or about 102 nucleic acids encoding same, respectively) of each NCSMECD polypeptide (or nucleic acid encoding said polypeptide) are absent.For example, in CD28BP-15 polypeptide (SEQ ID NO:66), the amino acidsequence of the signal peptide comprisesMGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSG (SEQ ID NO:315) (see FIG. 2A), andthe nucleic acid sequence that encodes the signal peptide comprises atleast about the first 102 nucleotide residues of SEQ ID NO:19. Thesignal peptide sequence of NCSM molecules may vary in length. One ofordinary skill in the art can readily determine the amino acid sequenceof an NCSM molecule that comprises the signal peptide sequence byaligning the full-length amino acid sequence of WT hB7-1 with thefull-length amino acid sequence of the NCSM molecule, comparing thesignal peptide sequence of WT hB7-1 with the corresponding sequence ofthe NCSM molecule, and determining the segment of the full-length aminoacid sequence of the NCSM molecule that corresponds to the signalpeptide. The nucleic acid sequence that encodes the signal peptide ofhB7-1 comprises the nucleotide residues of SEQ ID NO:273 that encode thesignal peptide of SEQ ID NO:278. The nucleic acid sequence that encodesthe signal peptide of an NCSM molecule can be similarly determined bycomparison of the full-length nucleic acid sequence of WT hB7-1 with thefull-length nucleic acid sequence of the NCSM molecule.

FIG. 14A shows a schematic representation of a hB7-1-ECD fused to anE-epitope amino acid sequence and a hexa-His tag (SEQ ID NO:299) aminoacid. The amino acid sequences corresponding to the E-epitope andhexa-His tag, and selected amino acids of the ECD, are shown.

A representative plasmid expression vector, termed pNSCMsECD, comprising6063 bps and encoding a soluble NCSM ECD, made by the above procedure isshown in FIG. 15. The vector includes, among other things, selectedelements of the pCDNA3.1(+) backbone, including an ampicillin resistantgene, A^(R), and a bovine growth hormone (BGH) poly A terminationsequence; nucleic acid sequence encoding an E-epitope/his tag; nucleicacid sequence encoding a NCSM-ECD; and a CMV promoter (e.g., known or WTCMV promoter, such as human CMV promoter, or recombinant or chimeric CMVpromoter). A plasmid vector encoding a soluble hB7-1-ECD or fragmentthereof can be made by substituting a hB7-1-ECD sequence or fragmentthereof for the NCSM-ECD sequence shown in the figure.

A plasmid expression vector encoding either a soluble NCSM-truncated ECD(e.g., NCSM ECD fragment) or a soluble WT hB7-1-trunECD can be madeusing the procedure above by substituting a truncated NCSM ECD nucleicacid sequence or truncated hB7-1 ECD nucleotide sequence for NCSM ECDnucleotide sequence. Table 6 shows the positions of nucleotide residuesand corresponding amino acid residues of the signal peptide sequence andexemplary truncated ECD domains of selected CD28BP and CTLA-4BP clonesand WT hB7-1 ECD, and the last three amino acid residues at the 3′ endof each truncated ECD of a NCSM clone or WT hB7-1.

Nucleotide Amino Acid Clone ID Position Position 3′ end ECD wild-typehuB7.1-ECD 1-702 1-234 NTT CD28BP-8 ECD 1-717 1-237 SKP CD28BP-11 ECD1-717 1-237 SKP CD28BP-15 ECD 1-717 1-237 SKP CTLA4-5X2-8C ECD 1-7021-234 NTP CTLA4-5X2-10C ECD 1-702 1-234 NTP CTLA4-5X4-1F ECD 1-702 1-234NTP CTLA4-5X4-11D ECD 1-702 1-234 NTP CTLA4-5X4-12C ECD 1-702 1-234 NTPCTLA4-5X5-2E ECD 1-702 1-234 NTP CTLA4-5X5-6E ECD 1-702 1-234 NTTCTLA4-5X6-9D ECD 1-702 1-234 NTP CTLA4-5X8-1F ECD 1-702 1-234 NTPNucleotide Amino Acid Clone ID Position Position 5′ end Fc X70421 HuIgG1-Fc 85–768 29–256 DKTH . . .

The invention also provides for ECD domains of the NCSM polypeptides(and nucleic acid sequences encoding such polypeptides) that lack thesignal peptide sequence; the first 34 amino acids (or 102 nucleic acidsencoding same) of each NCSM ECD polypeptide (or nucleic acid encoding aNCSM ECD polypeptide) are absent. In secreted forms, the signal sequenceis cleaved.

B. Expression and Purification of Soluble ECD and NSCM Polypeptides.

Soluble polypeptides from the CTLA-4BP-ECD, CD28BP-ECD, and hB7-1-ECDexpression vectors described above was expressed by transfection ofthese vectors into cells of a human HEK 293 cell line using SuperfectReagent (Qiagen) and expression of the sequence encoding the NCSM ECD ora fragment thereof. In each case, soluble protein was purified fromcrude culture supernatants using a Hi-Trap anti-e-epitope mAb affinitycolumn (Amersham-Pharmacia, Piscataway, N.J.) followed by bufferexchange into PBS, according to the manufacturer's instructions. Purityof a recovered fusion protein was assessed by SDS-PAGE followed byeither coumassie stain or immunoblotting with a mouse anti-penta-His mAb(Serotech, UK), performed according to manufacturer's instructions andusing known methods as described in, e.g., Rapley and Walker; Harlow andLane; Colligan; Sambrook, all supra (data not shown). The SDS-PAGEresults for soluble hB7-1-ECD and soluble CD28BP-15 sECD revealed amolecular weight (MW) of ˜50 kDa for each, as shown in FIG. 16. Areference mixture spotted in the far-left lane indicated bands ofcompounds of known MWs of 188 kiloDaltons (kDa), 98 kDa, 56 kDa, and 31kDa, respectively, for comparison. SDS-PAGE analysis showed a hB7-1-Igfusion protein homodimer of ˜140 kDa; this dimer is believed to containa covalent linkage between cysteine residues of the hinge-CH2-CH3 domainof the Fc. A hB7-1-Ig monomer thus has an apparent MW of ˜70 kDa. A˜70-kDa monomer of hB7-1-Ig-delta Cys mutant fusion protein was alsoobserved (see FIG. 16). It is believed the deleted cysteine (δCys)mutant prevents covalent dimerization of two individualB7-1-ECD/hinge-CH2-CH3 (Ig) molecules.

C. Expression Vectors Encoding Soluble NCSM-Ig Fusion Proteins.

Mammalian expression plasmids encoding a soluble NCSM-ECD-Ig fusionprotein and soluble WT hB7-1-ECD-Ig fusion protein were constructedfirst by PCR amplification using pfu turbo polymerase (Stratagene, LaJolla, Calif.) of selected NCSM ECDs or hB7-1-ECD from plasmidscontaining a full-length NCSM DNA sequence and hB7-1 DNA sequence asdescribed above. The PCR primers were designed to anneal specificallywith the first or last 20–24 nucleotides of a particular nucleic acidregion corresponding to the ECD of a specific NCSM or hB7-1, and flankedby restriction sites BamHI and BsteII, at their 5′ and 3′ ends,respectively. In some instances, the small peptide linker forming thein-frame translational coupling between the NCSM ECD (or hB7-1) and theIgG1 Fc contained the sequences valine-threonine (VT) orglycine-valine-threonine (GVT), depending upon the nucleotide sequencecompatibility of the 3′ codon of the NCSM ECD. Incorporation of theBsteII restriction site at the fusion junction creates the in-framevaline-threonine linker. The factor Xa cleavage site (IEGR) was insertedbetween the 3′ end of the NCSM ECD (or hB7-1 ECD) and 5′ end of the GVTor VT linker to allow production of sECD void of the Fc domain (FIG.14B).

DNA sequences encoding human IgG1 Fc were obtained from human spleenmRNA (Clontech, Palo Alto, Calif.) using a RT-PCR kit (Stratagene, LaJolla, Calif.) according to the manufacturer's instructions, and primersspecific to the first or last 20–28 nucleotides of the sequencecorresponding to the entire human IgG1 Fc hinge domain or a fragmentthereof (see, e.g., the protein sequences shown at GenBank AccessionNos. P01857 and X70421, respectively; other Fc sequences can also beused) flanked by restriction sites BstEII and EcoRI, at their 5′ and 3′ends, respectively (FIG. 14B). Alternatively, a variant derived from ahuman IgG1 Fc hinge domain (e.g., GenBank Access. No. P01857 or X70421)can be prepared that imparts specific desirable biological andpharmacological properties to the soluble NCSMs.

The IgG1-Fc amplicon (˜730 bp) was digested with BstEII and EcoRI andsubsequently cloned into pCDNA3.1(+) expression vector (serving as abackbone vector) digested with BamHI and EcoRI. The NCSM-Ig expressionplasmids were constructed by ligating (fusing in-frame) low melt agarosepurified NCSM BamHI-BstEII DNA fragments (˜730 bp), Ig-Fc BstEII-EcoRIDNA fragments (˜730) and pCDNA3.1(+) BamHI-EcoRI DNA fragment (˜5400 bp)to produce soluble NCSM-ECD-IgFc fusion expression vectors (see FIG.17).

A sequence complementary to either that corresponding to the sequenceshown at GenBank Accession No. X70421 or P01857 and an IgG Fc variantcontaining two amino acid substitutions (D234E and L241M) were clonedand deduced by DNA sequence analysis using known methods. The NCSM-Igfusions proteins may contain an IgG Fc sequence corresponding to thatshown at GenBank Accession No. X70421 or P01857 or IgG Fc variantsequence as the fusion partner.

FIG. 14B shows a representation of a soluble WT human B7-1-ECD-Igsequence, including the signal domain, ECD, Factor Xa (IEGR (SEQ IDNO:300)), VT or GVT linker, and human B7 hinge CH2-CH3 domain of the Fcregion of IgG1 corresponding to the sequence shown at GenBank AccessionNo. P01857. The amino acid residues positioned at the beginning and endof a representative ECD domain and 5′ end of the human B7 hinge CH2-CH3domain are shown. A NCSM-ECD-Ig sequence would be comparable to thatshown for hB7-1ECD-Ig in FIG. 14B.

Nucleotide sequences encoding truncated NCSM ECDs or truncated hB7-1ECDswere also used to make NCSM-trunECD-Ig and hB7-1-trun ECD-Ig expressionconstructs and fusion proteins, respectively. Truncated NCSM ECDstypically contained at least one less amino acid residue than thefull-length NCSM ECD; nucleotide sequences encoding truncated NCSM ECDscomprised corresponding fewer nucleotides.

The nucleotide positions and corresponding amino acid positions of anexemplary full-length ECD or truncated ECD of selected NCSM clones andhB7-1 used for construction of expression plasmids encoding the fusionproteins are shown in Tables 5 and 6. Table 5 shows the nucleotidepositions and corresponding amino acid positions of the hIgG1 Fcsequence shown at GenBank Accession No. P01857, and amino acid residuesat the 5′ end of this Fc region. Table 6 shows the nucleotide positionsand corresponding amino acid positions of the hIgG1 Fc sequence atGenBank Accession No. X70421, and amino acid residues at the 5′ end ofthis Fc region. The invention also provides for fusion proteins in whichthe ECD domains of the NCSM polypeptides (and nucleic acid sequencesencoding such polypeptides) lack the signal peptide sequence; in thisaspect, the first 34 amino acids (or 102 nucleic acids encoding same) ofeach NCSM ECD polypeptide (or nucleic acid encoding NCSM ECDpolypeptide) is absent. In secreted forms, the signal sequence iscleaved.

A representative plasmid vector encoding a soluble hB7-1-ECD-Ig fusionprotein, phB7-1-ECD-Ig, is shown in FIG. 17. The vector includes, amongother things, selected elements of the pCDNA3.1(+) backbone, includingan ampicillin resistant gene, A^(R), and a bovine growth hormone (BGH)poly A termination sequence; nucleic acid sequences encoding ahB7-1-ECD/IgG1 Fc fusion protein; and a CMV promoter (e.g., known or WTCMV promoter, such as human CMV promoter, or recombinant or chimeric CMVpromoter). A plasmid vector encoding a soluble NCSM-ECD-IgG1 fusionprotein or fragment thereof can be made by substituting a NCSM-ECDsequence or fragment thereof for the hB7-1 ECD sequence in the vectorshown in FIG. 17. Plasmid vectors encoding a hB7-1-trunECD-Ig or NCSMtrunECD-Ig fusion protein are also be made using the same procedure.

In another aspect, a non-dimerizing Ig-Fc domain (PKSCDKTHTCPPCP (SEQ IDNO:298)→PKSSDKTHTSPPSP (SEQ ID NO:301)) was engineered by PCRmutagenesis (Stratagene, La Jolla, Calif.) to mutate the cysteineresidues within the Ab hinge region to serine residues so as to preventthe formation of NCSM-ECD-Ig or hB7-1-ECD-Ig homodimers covalentlylinked by disulfide bonds between the hinge-CH2 cysteines of neighboringNCSM-ECD-Ig or hB7-l-ECD-Ig molecules. This non-dimerizing Ig-Fc domaincan alternatively be used as the Ig portion in an NCSM-ECD-Ig orhB7-1-ECD-Ig fusion protein prepared as described above. Affinitypurified huB7-1-ECD-IgδCys, comprising hB7-1-ECD fused to Ig in whichthe cysteines were mutated (represented by delta or δCys) was shown tohave a molecular weight of ˜70 kDa (molecular size of non-disulfidelinked Fc fusion monomer) (FIG. 16). The present invention providessimilarly prepared Cys-mutant Ig fusion proteins, NCSM-ECD-IgδCys orNCSM-trunECD-IgδCys, and nucleic acid sequences encoding such proteins.

D. Expression and Purification of Soluble NCSM ECD-Ig Protein

Soluble protein from CTLA-4BP-ECD-Ig fusion and CD28BP-ECD-Ig fusionexpression vectors was expressed by transfection into cells of a humanHEK 293 cell line using Superfect Reagent (Qiagen) and purified fromcrude cultured supernatants using a Hi-Trap Protein-A affinity column(Amersham-Pharmacia, Piscataway, N.J.) followed by buffer exchange intoPBS, according to the manufacturer's instructions. Purity of therecovered fusion protein was assessed by SDS-PAGE followed by eithersilver stain or immunoblotting with a goat anti-human IgG Fc specifichorseradish peroxidase (HRP) mAb (Kirkegaard and Perry Laboratories),according to manufacturer's instructions and known methods as describedin, e.g., Rapley and Walker, Sambrook, and Colligan, all supra.

As an example, a soluble wild-type human B7-1-ECD-Ig fusion protein(e.g., comprising, in part, an ECD fragment, “truncated ECD,” orfull-length ECD of hB7-1) was purified from human HEK 293 cellstransfected with the expression plasmid, phuB7-1 ECD-Ig, using Protein-Aaffinity chromatography and fractionated on a SDS-PAGE. Serial dilutionsof soluble human B7-1 ECD-Ig fusion protein showed a band whichco-migrated with a commercially available form of soluble WT humanB7-1-ECD-Ig or B7-2-ECD-Ig fusion protein from R&D Systems, thusdemonstrating that a protein of the correct molecular weight for asoluble WT hB7-1-ECD-Ig fusion protein was produced in vitro (data notshown). The results indicated that WT hB7-1 ECD-Ig fusion protein ispredominantly an ECD-Ig homodimer fusion protein. (Soluble hWT hB7-1 ECDmonomer was shown to have a molecular weight on SDS-PAGE of about ˜50kDa.) SDS-PAGE analysis revealed the affinity purified CD28BP-15 ECD-Igand CTLA-4BP 5x4-12C ECD-Ig fusion proteins co-migrated with WT humanB7-1 ECD-Ig fusion proteins, as shown in FIG. 18, and thus havemolecular weights nearly identical to or at least approaching that of WThB7-1 ECD-Ig fusion protein. A reference mixture included at thefar-left shows bands of compounds of known MWs (FIG. 18).

Crude supernatants from HEK 293 cells transfected with expressionplasmids encoding either a soluble (e.g., truncated or full-length ECD)fusion protein form of various CTLA-4BPs and CD28BPs of the invention, aWT hB7-1-ECD-Ig, or a pCDNA3.1(+) vector control were fractionated on anSDS-PAGE, blotted to nitrocellulose and hybridized with a goatanti-human IgG Fc specific HRP mAb using known Western blotting methods(e.g., Rapley and Walker; Sambrook; Harlow and Lane, all supra).Results, shown in FIG. 19, showed a predominant band and fainter bandaround ˜140 kD and ˜70 kDa, respectively, which correspond to thepredicted molecular weight of dimeric and monomeric forms of the NCSMfusion proteins. 8 potential N-glycosylation sites located in the ECD.Note that the MWs are approximate weights, since the proteins may beglycosylated. A band co-migrating with soluble WT hB7-1ECD-Ig wasvisible for all CTLA-4BP ECD-Ig and CD28BP ECD-Ig fusions.NCSM-trunECD-Igs also showed similar results to hB7-1-trunECD-Igs. Thesupernatant from the negative control transfection (HEK 293 cellstransfected with a pCDNA3.1(+) vector) did not produce a detectableband. Oligomeric or multimeric forms (NCSM-ECD-Ig dimers, trimers, etc.)were observed for CTLA-4BPs (Clones 5X4-11D, 5X4-12C, 5X5-2E, 5x 8-1F)and CD28BPs (Clones 8 and 11).

E. Construction of Stable Cell Lines Expressing Soluble NCSM-ECD andNCSM-ECD-Ig

Stable 293 cell lines expressing soluble NCSM-ECD (NCSM-sECD)polypeptides or soluble NCSM-ECD-Ig fusion proteins were produced byelectroporating HEK 293 cells with DraIII digested expression plasmidscomprising nucleic acid sequences encoding such polypeptides or proteinsaccording to the known methods as described in, e.g., Ausubel andSambrook, both supra. Stable integrants were selected using DMEMcontaining 10% FCS and 2 mg/ml G418 antibiotic (Geneticin, Gibco-BRL).Purification was performed as described above except the eluatecontaining NCSM-Ig fusions from the Protein-A column was furtherpurified by standard gel-filtration chromatography (size-exclusionchromatography) (see, e.g., Rapley and Walker; Sambrook, both supra)using a Superdex 200 10/30 (24 ml) column (Amersham-Pharmacia)(following manufacturer's instructions) to remove non-NCSM proteins. Theapproximate apparent molecular weights (App MW) of purified solubleNCSMs as determined by this gel-filtration analysis are shown in Table 7below.

TABLE 7 Molecule Type App MW (kDa) sCD28BP-15-ECD  ~49.04 HuB7-1/IgFc(commercial, R&D Systems) ~643.39 wtHuB7-1-Ig ~320.02 wtHuB7-1-IgδCys~403.31 CTLA-4BP 5X4-12C-ECD-Ig ~330.28 CTLA-4BP 5x4-12C-ECD ~106  CD28BP-15-ECD-Ig ~345.54

In addition to monomers of NCSM-ECD-Ig and NCSM-trun-ECD-Ig, the presentinvention includes aggregates and multimers (e.g., crosslinked forms) ofthe soluble NCSM polypeptides of the invention, such as, e.g.,NCSM-ECD-Ig and NCSM-trun-ECD-Ig, where the Ig portion comprises an Fcregion or variant thereof as described above.

F. In Vitro Characterization of Biological Activities

1. T Cell Proliferation Assays Using Soluble NCSM Fusion Proteins

Soluble NCSM were generated and purified as described above. Humanwild-type B7-1 was also expressed using the same methods. In addition, acommercial human wild-type B7-1-Ig fusion protein was obtained from R&DSystems (see also Table 7). To characterize the biological properties ofthese molecules, two different formats were used to further analyze theeffects of crosslinking and non-crosslinking on the function of themolecules. More specifically, we analyzed the fusion proteins both asstandard non-crosslinked soluble molecules as well after preincubationwith mAbs specific for the Fc portion of human IgG (crosslinkedmolecules). Crosslinking has previously been shown to affect thefunction of wild-type human B7-1 (Rennert et al., Intl Immunol. 1997June;9(6):805–13). Crosslinked molecules comprise at least two moleculesof interest, e.g., multimers, such as two NCSM molecules. Anon-crosslinked NCSM molecule comprises one NCSM molecule.

Purified human T cells were used in these studies. T lymphocytes weresorted using a Moflow flow cytometer by the methods described previouslyin “T Cell Proliferation Assay” in the “Materials and Methods” sectionabove and used at 1×10⁵ cells/well in U-shaped 96-well assay plate. Tcells were cultured in the presence of soluble anti-CD3 mAbs(Pharmingen, San Diego, Calif.) to deliver a primary signal (also calledSignal 7). The secondary signal to T cells was delivered by adding thepurified Ig fusion proteins; e.g., WT hB7-1-ECD-Ig, CD28BP-ECD-Ig, orCTLA-4BP-ECD-Ig were added at various concentrations, and anti-CD28 mAbs(Pharmingen) were used as a positive control. To obtain crosslinkedIg-fusion molecules, purified Ig fusion proteins were pre-incubated with5-fold excess of affinity-purified goat anti-human IgG Fc portion (KPL,Gaithersburg, Md.) for 30 minutes (min) on ice prior to use. Thecross-linked complex was then added into 96-well plate containing Tcells in total volume of 200 ul of Yssel's medium (Yssel et al. (1984) JImmunol Methods 72(1):219) supplemented with 10% FBS. Assay plates wereincubated for total of 3 days. The cultures were pulsed with 1microCi/well of ³ H-thymidine (Amersham, Piscataway, N.J.) during thelast 8 hours of incubation and then harvested. ³H-thymidineincorporation (cpm) was calculated from triplicate cultures.

A representative experiment using crosslinked fusion proteins andpurified human T cells is shown in FIG. 20A. Increasing concentrations(conc) of soluble Ig-fusion proteins of hB7.1 (solid square), CD28BP-15(open triangle) and a control antibody human IgG (open circle) wereadded at various concentrations to the cultures as indicated. A fixedconcentration (125 micrograms/milliliter (25 ug/ml)) of goat anti-humanIgG Fc was preincubated with soluble Ig-fusion proteins prior to use.The data represent a mean+/−SD of C.P.M. Both crosslinkedCD28BP-15-ECD-Ig-fusion protein and WT hB7-1-ECD-Ig fusion proteininduced a strong proliferative effect on purified human T cells culturedin the presence of anti-CD3 mAbs. CD28BP-15-ECD-Ig fusion proteininduced a more significant T cell proliferation response than didhB7-1-ECD-Ig fusion protein after cross-linking with goat anti-human IgGFc antibodies. FIG. 20E represents a summary of data from 4 separateexperiments using crosslinked fusion proteins comprising NCSM multimersand purified human T cells as described in FIG. 20A above. The datarepresent a mean+/−SEM of C.P.M. Based on these analyses usinganti-human IgG mAbs to make multimeric, crosslinked NCSM molecules,crosslinked CD28BP-ECD-Igs (e.g., CD28BP-15-ECD-Ig) promoted activationof the CD28 receptor to a greater extent than did crosslinked WThB7-1-ECD-Ig.

These data indicate that the formulation/multimerization of the solubleNCSM fusion proteins and soluble NCSMs significantly affects theirbiological properties. That is, soluble multimeric forms of NCSMmolecules (e.g., comprising two or more subunits of a NCSM-ECD-Ig,NCSM-trunECD-Ig, NCSM-ECD, NCSM-trunECD or other protein and fusionprotein variants thereof) have biological properties that are differentfrom corresponding soluble monomeric forms. In such multimeric forms,each NCSM subunit of the multimer need not be identical. Solublemultimeric forms comprising two or more NCSM subunits (e.g., NCSN-ECDsubunits) can be made by crosslinking, using leucine zippers, orengineering the subunits by other means known to those skilled in theart for making multimers or aggregates. When tested as soluble moleculeswithout crosslinking, soluble monomeric CD28BP-15-ECD-Ig fusion proteinsinhibited PHA-induced proliferation of human PBMC (see below). However,similar to the membrane-bound version of CD28BP-15 (full length), thecrosslinked soluble CD28BP-ECD-Ig molecule strongly enhanced theproliferation of purified T cells in the presence of soluble anti-CD3mAbs. These data indicate that crosslinked or aggregated (e.g.,multimeric) forms of soluble forms of the NCSM polypeptides (e.g.,soluble NCSM-ECD-Ig, NCSM-trunECD-Ig, NCSM-ECD, NCSM-trunECD and otherprotein and fusion protein variants thereof) are promising drugs toactivate the immune system, which is expected to be beneficial in thetreatment of malignant diseases (e.g., cancer), infectious diseases, andimmunodeficiencies. The crosslinking can be generated in vitro (e.g., asdescribed in assays above) or in vivo (e.g., through high-affinitybinding to Fc receptors on antigen-presenting cells). In addition, whenusing cell-based vaccines, the crosslinking can be caused bytransfecting receptors for human IgG (Fc receptors) into the cells thatare used as vaccines (and the NCSM-Ig fusion binds to the Fc receptorsexpressed on the cells that are used as vaccines. In contrast, withoutprior crosslinking, the soluble NCSM polypeptides inhibited thePHA-induced proliferation of human PBMC, indicating that they haveinhibitory effects on human T cell function. Furthermore, solubleCD28BP-ECD (without Ig portion) strongly inhibited T cell proliferationin vitro. Therefore, these soluble NCSM polypeptides are promising drugsfor the treatment of autoimmune and inflammatory diseases, such asrheumatoid arthritis, multiple sclerosis, inflammatory bowel disease,psoriasis, and organ transplantation.

When PBMCs were cultured in the presence of the soluble molecules ofCD28BP-15-ECD-Ig or CTLA-4 BP 5x4-12c-ECD-Ig without prior crosslinking(and no PHA), no T cell activation was observed except in the case ofinsect cell derived commercial wild-type B7-1-Ig (see Table 7). Anincreasing concentration of non-crosslinked ECD-Ig fusion proteins ofhB7.1 (solid square), and a control antibody human IgG (open circle),and commercially obtained insect cell derived human B7-1-Ig fusionproteins (R&D Systems) (solid triangle), were added to cultures of PBMCand proliferation was measured as described above (FIG. 20B). The insectcell derived human B7-1-Ig fusion proteins induced proliferation ofhuman PBMC, whereas the hB7-1 expressed in 293 cells did not induce Tcell proliferation. When these proteins were analyzed by gel filtration,it was evident that the protein expressed in insect cell had a 2-foldhigher molecular weight than hB7-1-Ig fusion expressed in 293 cells(Table 7). These data further support the conclusion that highermolecular weight aggregates (e.g., crosslinked or multimeric molecules)improve the capacity of these soluble NCSM polypeptides to signalthrough their respective ligands.

We also performed a PHA-activated T cell proliferation assay. In thisassay, PBMCs were isolated from human blood by centrifugation overHistopaque-1077. PBMCs were used at 1×10⁵ cells/well in 96-well roundbottom plate (Costar); each well contained a total volume of 200 ulYssel's medium (Yssel et al. (1984) J Immunol Methods 72(1):219)supplemented with 10% FBS. 1 ug/ml of PHA (Phytohemagglutinin) (Sigma,St. Louis Mo.) was added into these cultures. PHA is a plant protein(lectin-like molecule) that is extracted from Phaseolus vulgaris (redkidney bean) and binds to various sugars and sugar residues inoligosaccharides. PHA is used as a T cell mitogen to activate T cells(including, e.g., memory T cells, naive T cells, etc.). Increasingconcentrations (ug/ml) of soluble non-crosslinked CD28BP-15-ECD-Ig (opentriangle), non-crosslinked hB7-1-ECD-Ig (solid square), R&DhB7-1-Igfusion proteins (R&D Systems, Minneapolis, Minn.) (solid triangle), madeas described previously, and CTLA-4-Ig ligand (R&D Systems) (soliddiamond) and control hIgG antibody (open circle)(Jackson ImmunoResearchLab. Inc., West Grove, Pa.) were added to these cultures as shown inFIG. 20C. The data represent mean+/−SEM of a representative of 4experiments, each preformed using triplicate wells. When the solubleCD28BP-15-ECD-Ig fusion proteins were cultured as soluble moleculeswithout prior crosslinking in the presence of PHA, they inhibitedPHA-induced proliferation of human PBMC in a dose-dependent manner (FIG.20C) and thus are useful in immunosuppressive applications. Wild-typehB7-1-ECD-Ig did not affect PHA-induced proliferation of human PBMCunder the same culture conditions (FIG. 20C). At most concentrations,CTLA-4-Ig inhibited PHA-induced proliferation of human PBMC more thandid hB7-1-ECD-Ig.

Furthermore, we studied the effect of soluble CD28BP-15-ECD (withoutIg-fusion) on PHA-activated PBMC. As shown in FIG. 20D, solubleCD28BP-ECD inhibited proliferation in a dose-dependent manner and theinhibition was greater than that induced by soluble hB7-1-ECD. Thesedata are in line with the conclusion that soluble CD28BP-15-ECD, incontrast to crosslinked soluble CD28BP-15-ECD-Ig, acts as an antagonistof CD28 signaling. In other words, the level of crosslinking andmultimerization determines whether soluble NCSM induce a positive signalthrough their receptors CD28 and CTLA-4, or whether they act asantagonists by binding to the receptors without significantly inducingactivation of the receptor (and thereby preventing the interaction ofthe endogenous ligands with these receptors). The effect of solublenon-crosslinked CD28BP-15-ECD-Ig fusion protein and CD28BP-15-ECD onmixed lymphocyte reaction (MLR), as described above, was studied usingpopulations of PBMCs from two human donors. PBMCs from one donor actedas a responder; PBMCs from the second donor, which were irradiated at2500 rads, served as a stimulator for the responder. Increasingconcentrations (ug/ml) of soluble non-crosslinked CD28BP-15-ECD-Ig (opentriangle), non-crosslinked hB7-1-ECD-Ig (solid square), non-crosslinkedCD28BP-15-ECD (solid triangle), non-crosslinked hB7-1-ECD (open square),and negative control hIgG antibody (open circle) were added to thesecultures as shown in FIG. 20F. These data represent an average of countsper minute (CPM) from 4 MLR experiments. CD28BP-15-ECD-Ig and CD28BP-ECDinhibited mixed lymphocyte reaction to a greater extent than didhB7-1-ECD-Ig and hB7-1-ECD.

G. Multimeric NCSM Molecules Using Leucine Zippers

Multimeric NCSM molecules are generated using leucine zippers. In thisapproach, leucine repeats are utilized to generate multimeric NCSMmolecules that have properties which are equivalent or substantiallyidentical to properties of the crosslinked NCSM multimers describedabove generated by crosslinking using goat anti-human IgG Fc mAbs (andcrosslinked NCSM fragments having such properties). The use of leucinezippers to oligomerize proteins is well known (see, e.g., Rieker and Hu(2000) Methods Enzymol. 323:282–96; Behncken et al. (2000) J. Biol.Chem. 275:17000–17007; Su et al. (1999) J. Immunol. 162:5924–5930).Leucine zippers comprise a motif comprising parallel α-helical coiledcoils as exemplified by those found in transcription factorsGCN4(RMKQLEDKVEELLSKNYHLENECARLKKLVGER) (SEQ ID NO:316), Fos(LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA) (SEQ ID NO:317), and Jun(RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN) (SEQ ID NO:318). The motif isrepresented by the occurrence of leucines in every seventh position andhydrophobic and branched amino acids occupying position 1 over four orfive heptad repeats. These motifs act as molecular clamps and facilitatehomo- and hetero- dimer formation of proteins. Homodimerization orhigher order self-oligomerization of a protein is also accomplishedusing other zipper motifs (e.g., Zhang et al. (1999) Current Biol.9:417–420) or mutated peptide sequences derived from transcriptionfactors, GCN4, Fos, or Jun.

Soluble monomeric NCSM molecules are modified with leucine zippers usingstandard molecular biology methods utilizing either natural (genomiceukaryotic/prokaryotic DNA or plasmid/viral DNA) or synthetic DNAencoding known or newly discovered oligomerization motifs. Thesesequence motifs are engineered as molecular zipper cassettes containingunique restriction enzyme sites to make in-frame fusion with either theN-terminus or C-terminus of a soluble monomeric NCSM. For example, a DNAsequence encoding the leucine zipper comprising the restriction site NotI at the 5′ end and restriction site Apa I at the 3′ end is used tofacilitate cloning into a vector backbone, such as, e.g., pCDNA3.1(GCGGCCGCa<GCN4>TAGGGGCCC (SEQ ID NO:319); GCN4 nucleotide sequence canbe codon optimized to improve translation in a particular cell ofinterest, such as a human cell). This allows for easy shuttling of DNAfragments encoding soluble monomeric NCSM-ECD polypeptides or monomericNCSM-ECD Ig-fusion polypeptides to oligomerization expression vectors.These expression vectors can also include an E-epitope and hexa-His tagfor diagnostic and purification of soluble proteins.

H. Variations of Soluble NCSM-ECD-Ig Fusion Proteins and Related NucleicAcid Sequences

Any of the NCSM polypeptide homologues of the invention are optionallyutilized in the construction of Ig fusion proteins and nucleic acidsencoding them. Full-length NSCM polypeptides of the invention can beused. Furthermore, various fragments of each NCSM polypeptide can beutilized in the construction of fusion proteins, including, e.g., theentire ECD of a NCSM polypeptide (such as CD28BP-15 or CTLA-4BP5x4-12c); various lengths or subsequences (e.g., truncated regions orfragments) of the ECD of a NCSM polypeptide; the cytoplasmic region of aNCSM polypeptide (and truncated regions and subsequences or fragmentsthereof); the transmembrane domain region of a NCSM (and truncatedregions and subsequences or fragments thereof), etc. Various additionalsequences can also be added to the NCSM-Ig fusion proteins, e.g.,various linker sequences (such as, e.g., Val-Thr), various proteolyticcleavage sites (such as, e.g., Factor Xa cleavage sites (IEGR),subtilisin, etc.), various Ig domains (or portions thereof), markers,purification sequences, restriction enzyme cleavage sites, and the like.As noted throughout, non-Ig sequences can also be fused to the givenNCSM sequences to produce fusion proteins.

For example, as illustrated above, NCSM polypeptide sequences of theinvention were utilized to construct Ig fusion proteins incorporatingboth linkers (V-T and G-V-T) and Factor Xa Cleavage sites. See, e.g.,FIGS. 14A–14B, Tables 5–6. The NCSM portions of these fusion proteinswere longer than the truncated NCSM sequences used to construct thefusion proteins as described elsewhere herein. Various sequence lengthsof NCSMs (both amino acid and nucleotide) can be utilized inconstructing Ig fusions as well as myriad, e.g., linkers and othersequences, etc. Various configurations of linkers, NCSM lengths, etc.are all aspects of the present invention. Any of the NCSM sequencesdescribed herein can be fused using essentially the same strategy.

The invention also provides nucleic acids encoding any of the variantsoluble NSMC polypeptides and fusion proteins described above orfragments thereof. Also included are vectors and expression cassettesincluding such nucleic acids.

Example V Construction of an Expression Cassette

A. Construction of Vector pMax Vax10.1

This example describes the construction of an exemplary vector forexpression in mammalian cells. The mammalian expression vector pMaxVax10.1 (see FIG. 21) comprises, among other things: (1) a promoter fordriving the expression of a transgene in mammalian cells;(2) apolylinker for cloning of one or more transgenes; (3) a polyadenylationsignal (polyA); and (4) a prokaryotic replication origin and antibioticresistant gene for amplification in E. coli.

1. Construction of minimal plasmid for amplification in E. coli.

The minimal plasmid Col/Kana comprises the replication origin ColE1 andthe kanamycin resistant gene (Kana^(r)). The ColE1 replication originmediates high copy number plasmid amplification. Alternatively, low copynumber replication origins, such as p15A (from plasmid pACYC177, NewEngland Biolabs Inc.) can be used.

The ColE1 origin was isolated by polymerase chain reaction (PCR) methodsknown in the art from vector pUC19 (New England Biolabs Inc.). To linkthe ColE1 origin to the Kanat^(r) gene, NgoMIV (or “NgoMI”) and DraIIIrecoginition sequences where added to the 5′ and 3′ PCR primers,respectively. NgoMIV and DraIII are unique cloning sites in the vector.For subsequent cloning of the mammalian transcription unit the 5′forward primer contains the additional restriction site NheI downstreamof the NgoMIV site and the 3 ′ reverse primer additional EcoRV and BsrGIcloning sites upstream of the DraIII site. All primers containadditional 6–8 base pairs overhang for optimal restriction digest. Thesequence for the 5′ forward primer is:acacatagcgccggcgctagctgagcaaaaggccagcaaaaggcca (SEQ ID NO:302). Thesequence for the 3′ reverse primer is:

aactctgtgagacaacagtcataaatgtacagatatcagaccaagtttactcatatatac (SEQ IDNO:303). The PCR reactions are usually performed with proof-readingpolymerases, such as Tth (PE Applied Biosystems), Pfu, PfuTurbo andHerculase (Stratagene), or Pwo (Roche), according to the manufacturer'srecommendations. A typical PCR reaction for Herculase polymerasecontains 1 μl template plasmid DNA (1–10 ng/μl), 5 μl 10× buffer, 1 μldNTPs (deoxynucleotide triphosphate) at 10 mM each, 1 μl forward primer(20 μM), 1 μl reverse primer (20 μM), 40 μl deionized, sterile water and0.5 μl Herculase polymerase in a 50 μl reaction. The PCR reaction isperformed at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 30 seconds per cycle, for a total of 25 cycles. The PCR productswere purified with phenol/chloroform using Phase lock Gel™Tube(Eppendorf) followed by standard ethanol precipitation. The purified PCRproducts were digested with the restriction enzymes NgoMIV and DraIIIaccording to the manufacturer's recommendations (New England Biolabs,Inc.) and gel purified using the QiaExII gel extraction kit (Qiagen)according to the manufacturer's instructions.

The Kanamycin resistant gene (transposon Tn903) was isolated by PCR fromplasmid pACYC177 (New England Biolabs, Inc.) using standard knownprocedures. The Kana^(r) gene is used for in vivo or in vitro studies.Alternative antibiotic resistant genes, such as ampicillin,tetracycline, and blasticidin resistant genes, can be used for in vivoor in vitro studies in a variety of cell cultures.

The 5′ PCR primers contain the DraIII cloning site and an additionalsingle restriction site, AscI, downstream of it. The 3′ PCR primerscontain the NgoMIV cloning site. The 5′ forward primer sequence is:ggcttctcacagagtggcgcgccgtgtctcaaaatctct (SEQ ID NO:304). The sequencefor the 3′ reverse primer is: ttgctcagctagcgccggcgccgtcccgtcaagtcagcgt(SEQ ID NO:305). The PCR reactions, product purification and digest withDraIII and NgoMIV were performed as described above. About 20 ng of eachof the two PCR products were ligated in a 20 μl reaction, containing 2μl 10× buffer and 1U ligase (Roche). Amplification in E. coli wasperformed using standard procedures as described in Sambrook, supra.Plasmids were purified with the QiaPrep-spin Miniprep kit (Qiagen)following the manufacturer's instructions and digested with BsrG1 andDraIII for subsequent ligation of the mammalian transcription unit(promoter and polyA).

2. Expression Vector pMax Vax10.1.

In this example, the CMV Towne promoter was used for driving theexpression of the transgene in mammalian cells. Alternatively, other CMVpromoters or non-naturally occurring recombinant or chimeric CMVpromoters can be used; for example, a chimeric or recombinant promoter,including an optimized CMV promoter, as described in copending, commonlyassigned PCT Application Serial No. US01/20123, entitled “Novel ChimericPromoters,” filed Jun. 21, 2001 as LJAQ Attorney Docket No. 02-031910PC,can be used, which is incorporated herein by reference in its entiretyfor all purposes. Different strains of CMV can be obtained from ATCC.Strains AD169 (VR-538; Rowe, W. (1956) Proc. Soc. Exp. Biol. Med.145:794–801) and Towne (VR-977; Plotkin, S. A. (1975) Infect. Immun.12:521–27) were isolated from human patients with CMV infections, whilestrains 68-1 (Asher, D. M. (1969) Bacteriol. Proc. 269:91) and CSG(Black, H. (1963) Proc. Soc. Exp. Biol. Med. 112:601) were isolated fromRhesus and Vervet monkeys, respectively. Other viral promoters, e.g.,from RSV and SV40 virus, and cellular promoters, such as the actin andSRα promoter, and the like, and other promoters known to those of skillin the art, confer ubiquitous transcription in mammalian cells as well.For cell type-specific transcription, the use of cell type-specificpromoters, such as muscle specific, liver specific, keratinocytespecific, and the like, and others known to those of skill in the artcan be used.

The CMV Towne promoter was isolated from DNA of the CMV virus Townestrain by commonly known PCR methods. The cloning sites EcoRI and BamHIwere incorporated into the PCR forward and reverse primers. The EcoRIand BamHI digested PCR fragment was cloned into pUC19 for amplification.For construction of the vector pMax Vax10.1, the CMV promoter wasisolated from the pUC19 plasmid by restriction digest with BamHI andBsrG1. The BsrG1 site is located 168 bp downstream of the 5′ end of theCMV promoter start, resulting in a 1596 bp fragment, which was isolatedby gel purified for subsequent ligation.

The polyadenylation signal from the bovine growth hormone (BGH) gene wasused in this example. Other poly A signals, which work well in mammaliancells, include, e.g., poly A signal sequences from, e.g., SV40, Herpessimplex Tk, and rabbit beta globin, and the like, and others known tothose of skill in the art. The BGH poly A was isolated from the pCDNA3.1vector (Invitrogen) using commonly known PCR methods. The 5′ PCR forwardprimer contained additional 14 bp sequence comprising recognition sitesfor the restriction enzymes PmeI and BglII, which form part of the polylinker. The 3′ reverse primer contains the restriction site DraIII forcloning to the minimal plasmid Col/Kana. The 5′ forward primer sequenceis: agatctgtttaaaccgctgatcagcctcgactgtgccttc (SEQ ID NO:306). The 3′reverse primer sequence is: acctctaaccactctgtgagaagccatagagcccaccgca(SEQ ID NO:307). The resulting PCR product was diluted 1:100, and 1 μlwas used as a template for a second PCR reaction with the same 3′reverse primer and a new 5′ forward primer. This primer was overlappingthe 5′ end of the template by 20 bp and contained another 40 bp 5′,containing BamHI, KpnI, XbaI, EcoRI and NotI recognition sequences toform the rest of the polylinker. The sequence of the 5′ extension primeris: ggatccggtacctctagagaattcggcggccgcagatctgtttaaaccgctga (SEQ IDNO:308). An alternative PCR product was generated with different 5′forward PCR primers to generate a vector with a modified polylinker,designated pMax Vax10.1mp (FIG. 21 with modified polylinker as describedabove). The orientation of the restriction sites in this polylinker is5′–3′: BamHI, XbaI, KpnI, EcoRI, NotI, BglII, and PmeI. The polylinkersequence is:ggatccactcatctagaacaatggtaccaatacgaattcggcggccgcagatctgtttaaacc (SEQ IDNO:309). The PCR products were digested with BamHI and DraIII and gelpurified.

The final ligation reaction contained about 20 ng each of the BsrG1 andBamHI digested CMV promoter, of the BamHI and DraIII digested polylinkerand BGH poly A, and the DraIII and BsrG1 digested minimal plasmidCol/Kana in a 50 μl reaction with 5 μl 10× ligase buffer and 2U ligase(Roche). Ligation, amplification and plasmid purification were performedas described above.

B. Construction of Vector pMax Vax with NCSM Polynucleotide Sequence

The nucleotide sequence encoding a NCSM polypeptide (e.g., a CD28BP orCTLA-4BP polypeptide or fragment thereof, such as an ECD domain) or anyother immunomodulatory molecule can be isolated by PCR with BamHI andKpnI restriction enzyme recognition sequences in the PCR forward andreverse primer as described above. In this example, a polynucleotidesequence encoding a CD28BP polypeptide (e.g., CD28BP-15 polypeptide (SEQID NO:19) is incorporated into the pMAx Vax 10.1 vector. To verify thecorrect sequence of the PCR products, the fragments are clonedconveniently into the TOPO® cloning vectors (Invitrogen) for sequencingaccording to the manufacturer's protocols. After BamHI and KpnIdigestion and gel purification, the genes are cloned into a mammalianexpression vector to confirm the expression of the gene. To clone thegenes into the polylinker of pMax Vax, the vector pMax Vax 10.1 mp (FIG.21 with modified polylinker as described above) was digested with BamHIand KpnI, gel purified and ligated to the respective genes, as describedabove. The construct pMax Vax-CD28BP (see FIG. 22A), which includes thenucleotide sequence encoding a CD28BP (here, e.g., SEQ ID NO:19), can beused for in vivo and in vitro expression in human and other mammaliancells and other cells in culture, including non-mammalian cells and thelike.

For in vitro expression the immune stimulatory molecules can also becloned in any commercially available vectors such, as pCDNA3.1+/−,pCDNA4 (Invitrogen), which are suitable for stable expression under drugselection in mammalian cells. If secretion is a desired feature, thegenes can be cloned into vectors such as pSecTag, pDisplay, pBC1(Invitrogen), which link the expressed proteins to secretion signals.For regulated expression vectors from the Tet™System (Clontech) orEcdysone regulatory vectors (Invitrogen) can be used. For highexpression levels in cell culture the immune stimulatory molecules canalso be cloned into viral vectors constructed from Retrovirus,Adenovirus (Clontec), and Sindbis virus (Invitrogen), or replicatingviral vectors constructed from EBV, BPV, HPV and SV40 virus. For in vivostudies, viral vectors constructed from Adenovirus, Lentivirus, andAlphaviruses, and the like can be used. If restriction sites other thanBamHI and KpnI are required for cloning into the different vectors,flanking restriction sites from the polylinker can be used.Alternatively, the genes can be isolated by PCR with the desiredrestriction sites located in the PCR primers as described above.

C. Bicistronic Vector pMax Vax-CD28BP-EpCAM/KSA

For immunotherapy studies it is desirable to express theimmunostimulatory molecule in the same cells as, for example, a cancerantigen. A nucleotide sequence encoding a cancer antigen, such asEpCam/KSA or a mutant or variant thereof, can be cloned into the pMaxVax vector (FIG. 21) to generate a pMax Vax-EpCam/KSA vector, using aprocedure analogous to that described above for cloning the CD28BPpolynucleotide sequence into the pMax Vax vector backbone. Twoexpression constructs, e.g., the pMax Vax-CD28BP vector (FIG. 22A) (orother pMax Vax-NCSM vector) and the pMax Vax-EpCam/KSA vector (or otherpMax Vax vector including a nucleotide sequence encoding an antigen),can then be co-transfected in cell culture or co-administered in vivo toa subject in need of such therapeutic or prophylactic treatment.

In an alternative format, which may be an optimal format for sometherapeutic or prophylactic applications, both the EpCam/KSA (or aEpCam/KSA mutant or variant thereof) and CD28BP genes (or a differentantigen gene and/or NCSM polynucleotide) can be expressed from the samevector. In one format, the resulting antigen and NCSM proteins can beco-expressed from a single promoter linked by an internal ribosomalentry site (e.g., IRES bicistronic expression vectors, Clontec). Thisexample describes the construction of an exemplary bicistronic vectorfor expression of at least one NCSM polypeptide and at least one antigenor antigen fragment (or a different co-stimulatory molecule) in whichthe NCSM polynucleotide and the nucleotide sequence encoding the antigenor antigen fragment form two separate expression units. In particular,this example describes the construction of a bicistronic vector forexpression of CD28BP (e.g., CD28BP-15) and the cancer antigen EpCAM/KSA(or mutant or variant thereof) in which the CD28BP polynucleotide andthe polynucleotide encoding the cancer antigen or antigen fragment formtwo separate expression units, each regulated by its own respectivepromoter and poly A signal. One of skill will understand that thisprocedure can also be readily adapted to construct a bicistronic vectorcomprising at least one NCSM polynucleotide of the invention (includingnucleic acid fragments thereof, and nucleic acids encoding soluble NCSMpolypeptides, peptide fragments thereof, and fusion proteins thereofdescribed herein) and a different antigen or antigen fragment (or adifferent co-stimulatory molecule).

The CD28BP gene is inserted into the polylinker of a pMax Vax vector asdescribed above, forming the first expression unit. The nucleic acidsequence of the cancer antigen, here the polynucleotide encoding theextracellular domain of EpCAM/KSA (or mutant or variant thereof), islinked to a second mammalian expression promoter (exemplary promotersinclude those set forth in this Example above and elsewhere) and asecond poly A signal (exemplary signals include those set forth in thisExample above and elsewhere) to form the second expression unit. In thisExample, a synthetic poly A (SPA) sequence was made and used. However,one of skill in the art would understand that other poly A sequences(e.g., bovine growth hormone (BGH) poly A or SV40poly A sequence) canalso be used. The synthetic poly A was derived from a sequence for therabbit β-globin poly A (Gen&Dev. 3:1019–1025 (1989). The sequencefragment was generated by annealing two oligonucleotides, whichcontained the respective cloning sites in the 5′ and 3′ sequences. Theupper strand sequence is: 5′-GATCTGTTTAAACTCTGGCTAATAAAAGATCAGAGCTCTAGACATCTGTGTGTTGGTTTTTTGTGTGTCTCACTCACAGA-3′ (SEQ ID NO:313), and the sequence of thelower oligonucleotide strand is: 5′-TGAGTGAGACACACAAAAAACCAACACACAGATGTCTAGAGCTCTGATCTTTTATTAGCCAGAGTTTAAACA-3″ (SEQ ID NO:314).

The second expression unit can be cloned into 3 different sites in theconstruct pMax Vax-CD28BP, both in forward or reverse orientation: (i)downstream of the first expression unit (e.g., CMV promoter-CD28BP-SPApolyA, CMVpromoter-CD28BP-BGH polyA, or CMVpromoter-CD28BP-SV40 polyA)using the single cloning sites DraIII and AscI in pMax Vax10.1; (ii)between the ColE1 and Kana^(r) gene using the single restriction sitesNgoMI and NheI; (iii) between the Kana^(r) gene and the CMV promoterinto the single EcoRV and BsrGI restriction sites (see vectordescription above in this Example). Independent of the location of thesecond expression unit it is advisable to add a terminator sequencedownstream of the first expression unit. A consensus terminator sequence5′-ATCAAAA/TTAGGAAGA3′ (SEQ ID NO:310) is described in Ming-Chei Maa etal. (1990) JBC 256 (21):12513–12519. In the construct pMax Vax,CD28BPthe sequence can be placed into the single DraIII site downstream of thepoly A sequence (e.g., synthetic poly A or SPABGH poly A sequence) (seeFIG. 22B).

This example describes the cloning strategy of the second expressionunit for location (ii). The second promoter (e.g., a WT CMV promoter,such as human CMV promoter or a recombinant CMV promoter with improvedexpression activity), the EpCAM/KSA cancer antigen (or mutant or variantthereof), and the second poly A (in the example, synthetic poly A orSV40 polyA), are isolated from the respective template plasmids by PCRor assembled from oligonucleotides (as described above in this Example).The PCR primers are designed to contain single restriction sites, whichallow for partial site-directed cloning of the three fragments into thefinal vector. The 5′forward PCR primer for isolation of the shuffled CMVpromoter contains the single NgoMIV (also called NgoMI) cloning site.The 3′reverse primer contains the NgoMIV site and another restrictionenzyme site, which does not cut in any of the other vector units (i.e.AccI, AgeI, AvrII, BsU361, MluI, RsrII, SalI) upstream of it separatedby a spacer of at least 10 base pairs. In the example AccI is chosen asthe additional cloning site. The PCR product is digested with NgoMIVfollowed by gel purification and cloned into the NgoMIV linearized andgel purified pMax Vax,CD28BP. The correct orientation of the second CMVpromoter after ligation is determined by PCR from bacterial colonies (asdescribed in Molecular Cloning, A Laboratory Manual, Sambrook andRussell) using the 3′ reverse primer and any forward primer of choicelocated about 500–600 bp upstream of the reverse primer in the CMVpromoter sequence. The second promoter containing plasmid is thendigested with AccI and NheI for cloning of the cancer antigen. The 5′primer for the EpCAM/KSA cancer antigen (or mutant or variant thereof)contains the single AccI site and the 3′ primer the single NheI site andan additional single restriction site upstream, AgeI, separated by aspacer of at least 10 base pairs. The PCR product is digested with theenzymes AccI and NheI and cloned into the equally digested vector. Theresulting construct is digested AgeI and NheI for cloning of the SV40polyA/terminator fragment. The 5′ forward primer for this PCR productcontains the single AgeI site and the 3′ reverse primer the terminatorsequence followed by the single NheI site. The 5′ cloning sequence andthe NheI site are incorporated in the oligonucleotides. The resulting(e.g., double-stranded) AgeI/NheI poly A fragment is then cloned in theequally digested vector. The cloning strategy is outlined below.

An exemplary construct pMax Vax,CD28BP,EpCAM/KSA is shown in FIG. 22B.

One of skill will understand that a similar procedure can be used toconstruct an expression vector comprising a nucleotide sequence encodinga CTLA-4BP of the invention (in place of the sequence encoding CD28BPabove in FIG. 22A. Such a vector can comprise a bicistronic vector, ifdesired, with a second nucleotide sequence of interest (e.g., encodingan antigen or another co-stimulatory molecule) included in the positionoccupied above by the antigen, as shown in FIG. 22B. One of skill willalso understand the above procedure can be readily adapted to constructan expression vector comprising different vector components, such asdifferent promoters, signal sequences, termination sequences,replication origin sequences, resistant gene or marker sequences.

Example VI Enhanced Immune Response Induced by a CD28BP Polypeptide

This example demonstrates the ability of a CD28BP molecule of thepresent invention (or fragment thereof) to enhance an immune response ofa heterologous antigen, such as a tumor-associated antigen (Ag), suchas, e.g., Ep-Cam/KSA (as described in Strand et al. (1989) Cancer Res.49:314–317; Szala et al. (1990) 87:3542–3546; Balzar et al. (1999) J MolMed 77:699–712), or a polypeptide variant, mutant, or derivative ofEpCam/KSA polypeptide (or a nucleotide sequence variant, mutant, orderivative encoding such EpCam/KSA polypeptide variant, mutant orderivative, respectively), or a pathogen antigen (e.g., hepatitis Bsurface Ag (HepBsAg)), in cynomolgus monkeys. The EpCam/KSA polypeptidevariant or mutant may comprise, e.g., an amino acid sequence ofEpCam/KSA in which at least one amino acid has been replaced by anotheramino acid; the substitution may comprise a conservative amino acidsubstitution. The corresponding nucleotide sequence may comprise, e.g.,a substitution of one or more nucleotide residues such that theEpCam/KSA polypeptide variant or mutant amino acid sequence is encodedtherefrom. In this example, a vector comprising a nucleotide sequenceencoding full-length clone CD28BP-15 is used. If desired, alternativelya vector comprising a nucleotide sequence encoding a fragment ofCD28BP-15 (e.g., such as an ECD) or encoding a fusion protein (e.g.,ECD-Ig) can be used. For example, a sequence encoding a soluble NCSM ofthe invention (e.g., CD28BP-15ECD, CD28BP-15-ECD-Ig, or with a trunECD,or the like) can be used. A vector comprising a nucleotide sequenceencoding a WT hepatitis B surface antigen (hepBsAg) (or fragmentthereof) and a vector comprising a nucleotide sequence encoding Ep-Camor EpCam variant or mutant (or a fragment of any of these) is used asthe antigen sequence. The procedure can be adapted to use any NCSMmolecule described herein and/or any antigen of interest, including,e.g. viral antigens or other cancer antigens described infra.

In the following example, separate vectors are prepared that encode eachof EpCam (or mutant or variant thereof), WT hB7-1, CD28BP-15, and theantigen. A separate control vector is also prepared. See Example V.However, as noted below and as described in Example V, a bicistronicvector encoding antigen (e.g., EpCam or a mutant or variant polypeptidesequence thereof) and B7-1, or encoding antigen (e.g., EpCam or mutantor variant polypeptide thereof) and CD28BP-15 can be used alternatively.If desired, a vector comprising a nucleic acid sequence encoding afragment of an NCSM polypeptide having the desired properties can beused, such as a nucleic acid sequence encoding a signal sequence (of SEQID NO:66 or another NCSM molecule or from B7-1) and the ECD of CD28BP-15(of SEQ ID NO:66), wherein the ECD has a CD28/CTLA-4 binding affinityratio that is at least about equal to or greater than that of B7-1and/or induces a T cell response that is greater than that induced byB7-1. Vectors comprising sequences encoding other antigens and/or NCSMmolecules, cytokines, costimulatory sequences, and the like or othervector elements can be constructed by using the vector constructionprocedures described above. One of skill will readily understand how tomodify/adapt these procedures to construct vectors comprising nucleotidesequences encoding such NCSM molecules with or without also encoding anyof such antigens.

In this analysis, five groups of cynomolgus monkeys (3 monkeys pergroup) are inoculated intradermally (i.d.) (e.g., by a gene gun orinjection with a needle) or intramuscularly using DNA plasmid expressionvectors with either CD28BP-15 alone, hB7.1 alone, antigen (Ag) alone(such as, e.g., EpCam/KSA or a mutant or variant thereof), CD28BP-15with Ag or hB7.1 with Ag, each at a total dose of 1 milligram DNA perinoculation as outlined in Table 8 below. For CD28BP-15, the vectortypically comprises a nucleotide sequence encoding SEQ ID NO:19 or anucleotide sequence encoding the polypeptide of SEQ ID NO:66. A DNAplasmid control vector lacking a nucleic acid insert encoding aCD28BP-15, hB7-1, or Ag is used to equalize the total amount of DNA usedin each injection. Procedures for constructing the pMax Vax plasmidvector alone (control vector) and a plasmid vector comprising anucleotide sequence encoding CD28BP-15 are described in Example V above.Similar procedures can be used to construct a separate pMax Vax vectoror the like comprising a nucleotide sequence encoding a human B7-1, Ag,or HepBsAg, as shown in Table 8. Alternatively, another plasmid-basedmammalian expression vector or a viral vector can be employed in thefollowing procedure, including any of those described above in thespecification. Immunized animals are monitored daily for any local andsystemic reactions.

TABLE 8 No. of Dose (mg DNA) Group animals Immunization for each vector1 3 CD28BP-15 vector + Control 0.5 + 0.5 vector 2 3 HB7-1 vector +Control vector 0.5 + 0.5 3 3 Ag vector + Control vector 0.5 + 0.5 4 3CD28BP-15 vector + Ag vector 0.5 + 0.5 5 3 HB7-1 vector + Ag vector0.5 + 0.5

Animals. Five groups of 3 male Cynomolgus monkeys, each weighingapproximately 4 kg (15 total), are used. Animals are randomly assignedto groups using a number draw. In addition, each animal is assigned aspecific number within that group. Inocula. Mixtures of plasmid DNA tocontain 0.5 mg of each (separate) vector component as outlined in Table8 are prepared. Total plasmid DNA delivered is 1 mg in each case. EachDNA expression plasmid is diluted in PBS, pH 7.4 from a stock solutionto achieve the target concentration in 1 ml per inoculum.

(Alternatively, a bicistronic format is used in which the followingplasmid vectors are made and substituted in the procedure: 1) a plasmidvector comprising a nucleotide sequence encoding EpCam or mutant orvariant polypeptide thereof (total DNA plasmid dose is 1 mg) (antigencontrol vector); 2) a plasmid vector comprising a nucleotide sequenceencoding both EpCam (or mutant or variant thereof) and WT hB7-1 (totalDNA plasmid dose is 1 mg) (antigen/WT hB7-1 control vector)(biscistronic vector that co-expresses Ag and hB7-1); 3) a plasmidvector comprising a nucleotide sequence encoding both EpCam (or mutantor variant thereof) and CD28BP-15 (or fragment thereof, includingsoluble form) (total DNA plasmid dose is 1 mg)(bicistronic vectorco-expressing EpCam(or mutant or variant thereof), and CD28BP-15). Thebicistronic vectors are prepared as described for the pMax Vaxbicistronic vector encoding both a CD28BP and EpCam (or mutant orvariant thereof) in Example V above.

Inoculation. Animals are anaesthetized prior to inoculation. The backsof the animals are first prepared by shaving the fur and the animals areinoculated by i.d. or i.m. injection with 1.0 ml of a total 1 mg DNAplasmid vector(s) as described in Table 8 above (or alternative amountsdescribed below) at multiple sites. The monkeys are boosted three timesat 3 weekly intervals with the same inoculation dose or with 0.1 mgAntigen protein (e.g., EpCam polypeptide or a polypeptide mutant orvariant thereof).

Observation and monitoring. Each inoculation site is examined every day,beginning at day 1, for any delayed-type hypersensitivity (DTH)reaction. Animals are observed daily for signs of systemic reaction tothe inoculation. These observations include, but are not limited to,changes in weight, body temperature, eating habits, skin and hair, eyes,mucous membranes, respiratory system, circulatory system, centralnervous system, somatomotor activity, elimination, behavior, and anyoccurrence of tremors, convulsions, salivation, diarrhea, lethargy, orcoma.

Collection of blood. Monkeys are bled to obtain 2–5 ml of whole bloodone day prior to immunization and weekly thereafter. Blood is allowed toclot, serum separated, frozen at −20° C. until further analysis. Onalternate weeks, however, 5–10 ml of blood is drawn in heparinized tubesfor T cell assay analysis.

Tissue collection. Punch biopsies of the inoculation site are takenaccording to standard known procedures once every three weeks.

Sample analysis. Antibody titers against each of Ep-Cam (or mutant orvariant thereof) and HepBsAg in the sera of the animals are determined,respectively, using ELISA assays (Mosolits et al. (1999) Cancer Immunol.Immunoth 47:315–320; Staib et al. (2001) Intl. J. Cancer 92:79–87; Chowet al. (1997) J. Virol. 71:169–178). Furthermore, T cell proliferationin response to one of these antigens is analyzed by adding 10 g/ml ofthe antigen to cultures of 10⁵ peripheral blood monocyte cells (PBMC).The cells are incubated for 3 days and incorporation of ³H-thymidineduring the last 8 hours of culture is measured by scintillation counting(as described in Punnonen et al. (1994) J. Immunol. 152:1094–1102). SeeT cell proliferation methods described above. A higher T cellproliferative response indicates a more vigorous immune response as aresult of the vaccination.

Cytokine production, such as, e.g., IFN-gamma, IL-2, IL-4, IL-5, IL-12,and IL-13 production, is studied in response to the specific antigenusing cytokine specific ELISAs (R&D Systems) or ELISpot assays(Biosource International, Camarrillo, Calif.), performed according tothe manufacturer's instructions. For example, enumeration of IFN-gammasecreting cells in single cell suspension is performed using a kitobtained from Biosource International (Camarrillo, Calif.) (seemanufacturer's instructions). The following protocol is used. 50 μl ofdiluted coating antibody is added to each well followed by the additionof 50 μl of PBS. Each well is incubated overnight at 4° C. Samples arethen aspirated and washed 5 to 10 times with wash buffer. 200 μl ofpost-coating solution is added into each well and wells are incubated 1hour at 37° C. or overnight at 4° C. The wells are aspirated and notwashed. Wells are 100 μl of prestimulated single cell preparation areadded into the wells. The plate is covered with the plate cover andincubated for 5 hours at 37° C. in a humidified atmosphere containing 7%CO₂. The wells are aspirated, 200 μl ice-cold deionized water is added,and the plate is placed for 10 min on melting ice. The wells are washed10 times with PBS. 100 μl of diluted biotinylated Antibody solution isadded, the plate is covered and incubated for 1 hour at 37° C. orovernight at 4° C. The wells are aspirated and washed 5 to 10 times withPBS. 50 μl of diluted-labeled anti-biotin antibody solution (GABA) isadded to each well. The plate is covered and incubated 1 hour at 37° C.The wells are aspirated and washed 5 to 10 times. 30 μl of activatorsolution is added to each well. The spot development is followed bylight microscopy. When clear spots have developed, the reactions arestopped by rinsing the wells with distilled water. The results arecompared between animals immunized with the antigen with or withoutCD28BP-15.

Such plasmid expression vectors encoding CD28BP-15 with and without anantigen are useful in therapeutic and prophylactic treatment protocolsas described above. Plasmid expression vectors encoding CD28BP-15 andEpCam/KSA (or mutants or variants of EpCam polypeptide or nucleotide)are useful in methods for therapeutically and/or prophylacticallytreating a variety of cancers, as described above. Given that theprimate model is an accepted model closely related to human, suchmethods may be readily adapted by one of ordinary skill in the art totherapeutic and/or prophylactic vaccination protocols for humans.

A similar procedure to that described above can be employed to assess anability of a CTLA-4BP of the invention to inhibit an immune response orinhibit T cell proliferation or CTL responses in a subject, bysubstituting a nucleotide sequence encoding a CTLA-4BP of the inventionin place of the nucleotide sequence encoding CD28BP-15 and using thefunctional assays for, e.g., T cell activation. For example, T cellactivation can be analyzed by measuring proliferation, cytokineproduction, CTL activity or expression of activation antigens such asIL-2 receptor, CD69 or HLA-DR molecules, as described above. Vectorsthat harbor CTLA4-BP genes that efficiently act through CTLA-4 areuseful in inducing, for example, tolerance and anergy of allergen- orautoantigen-specific T cells. In some situations, such as in tumor cellsor cells inducing autoimmune reactions, the antigen may already bepresent on the surface of the target cell, and the vectors encodingCTLA-4BP molecules may be transfected in the absence of additionalexogenous antigen gene.

Boosting. In methods described herein using either separate vectorsencoding each of Ag, hB7-1 or CD28BP, or bicistronic vectors encoding Agand CD28BP, or Ag and hB7-1, one or more additional doses of DNA plasmidvector (e.g., 1 mg) can be administered subsequently to an animal at oneor more subsequent intervals (e.g., 2 times), respectively enhance or“boost” the immune response. If desired, following a boosting of theimmune response with such administration of one or more additional DNAplasmid vector doses, at least one dose of the EpCam protein (proteindose of from about 0.1 to about 1 mg) (“protein boost) (or EpCam mutantor variant) can be administered to an animal to further enhance or“boost” the immune response. Additional protein boosts can beadministered subsequently at desired intervals (e.g., after one or moredays, one or more weeks, one or more months).

Example VII Blocking Development of EAE

The mouse model of Experimental Autoimmune Encephalomyelitis (EAE) hasmany similarities with human multiple sclerosis (MS), and it has beenwidely used as a model of human MS (see, e.g., Alvord, G. C. Jr., ed.,Experimental Allergic Encephalomyelitis: A Useful Model for MultipleSclerosis, Liss, NY (1984)). EAE can be induced in SJL/F mice by myelinbasic protein (MBP) or proteolipid-protein (PLP) or peptides thereof.

To demonstrate the efficacy of a CTLA-4BP molecule of the invention toprevent EAE, the following prophylactic treatment vaccination protocolis used. DNA expression plasmids encoding either CTLA-4BP or MBP (orPLP) are codelivered, or the two genes for CTLA-4BP and MBP (or PLP) arecoexpressed in the same vector and delivered, as follows. In thisexample, a nucleotide sequence encoding clone CTLA-4BP 5x4 12c is used.Procedures for constructing the pMax Vax plasmid vector alone (controlvector) or with a nucleotide sequence encoding a CD28BP and/or a secondpolypeptide (EpCam or mutant or variant thereof) are described inExample V above. One of skill can readily adapt such procedures toconstruct pMax Vax vectors comprising the nucleotide sequence encoding aCTLA-4BP and/or MLP (or PLP), expressed alone on separate vectors orcoexpressed on one vector. Alternatively, another plasmid-basedmammalian expression vector or a viral vector can be used in thefollowing procedure, including any of those described above in thespecification. 100 μg of the DNA plasmid in 100 μl PBS is injectedintramuscularly or intradermally to SJL/F female mice. A control DNAplasmid lacking the CTLA-4BP, MBP, or PLP nucleotide sequence issimilarly administered to a control group of mice.

To induce EAE, mice are injected intradermally with 100 μl rabbit brainmyelin basic protein (MPB) at 1 mg/ml in complete Freund's adjuvant.Mice are analyzed for the onset of EAE by visually noting tail paralysisfollowed by hind leg paralysis (at which point animals are sacrificedfor humane reasons).

The ability of a DNA plasmids encoding CTLA-4BP and/or MBP (or PLP) toblock EAE is demonstrated by the number of mice developing EAE and theseverity of the disease, as compared to mice that received the controlDNA plasmid.

Example VIII Improved Cell-based Vaccines for the Treatment of Cancer

To enhance the immunogenicity of tumor cells used as cell-based vaccinesfor the immunotherapeutic or prophylactic treatment of a variety ofcancers, patient tumor cells can be transfected with a CD28BP nucleicacid (NA) sequence of the present invention. In this example, thesequence corresponding to clone CD28BP-15 is used; however, other NAsequences of the invention can be readily employed. As an example, thespecific immunotherapy involves immunization of melanoma patients with apolyvalent, irradiated whole cell melanoma cells transfected with a DNAplasmid encoding CD28BP-15.

In one such method, a population of tumor cells derived from a melanomapatient's melanoma tumor cell lines (i.e., cells removed from thepatient) are transfected (e.g., by electroporation) with a sufficientlyeffective amount of DNA expression plasmid vector, pMax Vax, encodingCD28BP-15 (or fragment thereof, e.g., CD28BP-15-ECD or expressed solubleCD28BP) that facilitates uptake and expression of CD28BP-15 polypeptideon the cells; the amount of DNA plasmid typically constitutes atherapeutically or prophylactically effective amount or dosage to treatthe melanoma cancer or prevent further development of the cancer. ThepMax Vax plasmid is described in example V above. Or, anotherplasmid-based mammalian expression vector, or viral vector, can be usedin this procedure, including those described herein and throughout.

These transfected tumor cells are inactivated by irradiation (50 gray)and cryopreserved for used as the cell-based vaccine. Prior to treatment(delivery to the patient), the cells to be used as vaccine are thawedand washed 3 times in phosphate-buffered saline; if desired, the cellsto be used as a vaccine are formulated as a composition with anexcipient, such as, e.g., a pharmaceutically acceptable excipient, e.g.,PBS. (In an alternative format, allogeneic melanoma tumor cells aretransfected with a sufficient amount of pMax Vax DNA plasmid vectorencoding CD28BP-15 (or a fragment thereof, e.g., CD28BP-ECD) forCD28BP-15 expression.) Transfected tumor cells encoding an effectiveamount of expressed CD28BP-15 (or a composition comprising suchcells)—either those derived from the specific patient's cell line orallogeneic cells—are injected intradermally into the specific patient inauxiliary and inguinal regions in escalating doses once every 2 weeksfor 3 months. The first and second injections of the vaccine comprise2×10⁶ cells, followed by 6×10⁶ cells for the third and fourthinjections, and then 18×10⁶ cells for the fifth and sixth injections.

Immune responses of each patient are analyzed by measuring the levels oftumor cell specific Abs and the level of T cell response against theantigen or antigenic fragment expressed on the tumor cells by analyzingT cell proliferation in response to tumor cell lysates and measuringdelayed type hypersensitivity (DTH) reaction. T cell response againstthe cancer antigen is analyzed using standard methods described above(see, e.g., Example VI). Levels of tumor cell specific Abs in thepatients' sera are measured by ELISA using standard protocols (seeColligan; Sambrook; Rapley and Walker, all supra). To analyze DTH, tumorcell lysates are injected intradermally into the back of patients.Responses are evaluated on days 1, 2, 4, and 7 after injection. The meandiameter of induration is calculated as (greatest diameter+perpendiculardiameter)/2. A positive response is defined as a mean diameter ofinduration of 5 mm. Four-millimeter punch biopsies of positive reactionsare performed on selected consenting subjects to analyze the phenotypeof infiltrating cells using flow cytometry (FACSCalibur flow cytometerand CellQuest software, BDIS) as described above. The single cellsuspensions are then stained anti-CD3, CD4, CD8, CD 14, and CD20monoclonal antibodies to measure the percentages of T cells, CD4+ Thelper cells, CD8+ cytotoxic T cells, monocytes and B cells,respectively.

Estimated statistical survival rates are analyzed by the non-parametricKaplan-Meier method (see Kaplan et al., J Am Stat Assoc (1958) 53:457)(e.g., using the statistical analysis software JMP (ver. 3.1 forMacintosh; SAS Institute Inc., Cary, N.C.)). The log-rank test is usedto determine the differences in survival of patients from subgroupsdefined by different levels of risk factors. Survival times are definedas the length of time a given patient remains alive after the diagnosisof metastatic disease to either a regional site (AJCC Stage IIIA), withregard to skin and soft tissue metastasis, or a distant site (AJCC StageIV).

Example IX B7-1 Polypeptide Variants with Altered Properties

Sequence analysis of CTLA-4BP molecules exhibiting preferential bindingto CTLA-4 relative to CD28 revealed that many such molecules showed asubstitution of another amino acid for tyrosine at the amino acidposition corresponding to amino acid position 65 of the full-lengthamino acid sequence of hB7-1 (SEQ ID NO:278). These data suggested theamino acid residue at this position played a different role in thebinding of these molecules to CTLA-4 than to CD28. A polypeptide variantof the hB7-1 polypeptide shown in SEQ ID NO:278 was made which compriseda substitution of histidine for tyrosine at position 65 of the aminoacid sequence of SEQ ID NO:278, where the amino acid position ismeasured from the N-terminus. Position 65 corresponds to position 31 ofthe mature domain of hB7-1 (SEQ ID NO:278), as measured from theN-terminus. Specifically, codon TAC of the nucleic acid sequence ofhB7-1 shown in SEQ ID NO:273, corresponding to the three nucleic acidresidues at positions 193–195 of SEQ ID NO:273, was mutated to codonCAC. Digestion of the expression plasmid encoding hB7-1 with Pm1 I andXcm I removed a 200 base pair (bp) fragment which included the aminoacid at position 65 encoding Tyr (Tyr65). Using standard molecularbiology cloning methods, this nucleotide fragment was replaced with acorresponding Pm1 I-Xcm I DNA nucleotide fragment (200 bp) from thenucleic acid sequence encoding CTLA-4BP 5x4-12c, which encoded histidineat the amino acid position corresponding to position 65 of hB7-1 (SEQ IDNO:278). The remaining amino acids encoded by the 200-bp fragment wereotherwise identical between hB7-1 and CTLA4BP 5x4 12c. The B7-1polypeptide variant (termed “hB7-1-Tyr65His polypeptide variant”)comprised the full-length sequence of SEQ ID NO:278 with a Tyr65Hissubstitution (mutation). Methods for detecting and/or measuringexpression, binding to CD28-Ig and/or CTLA-4-Ig, and T-cellproliferation were performed as described above in previous Examples.

FIGS. 23A–23B show that the hB7-1-Tyr-65His polypeptide variant binds tosoluble CTLA-4-Ig more preferentially than it does to soluble CD28-Ig.FIGS. 23A–23B are histograms depicting the differential binding offull-length hB7-1-Tyr65His variant, CTLA-4BP 5x4-12c (gray histogram),and hB71 gray histogram) to either soluble CD28-Ig or CTLA-4-Ig. Thebinding levels are analyzed by flow cytometry (FACS) as describedpreviously. HEK 293 cells (2×10⁵ cells) transfected with either thenucleic acid sequence encoding hB7-1 (SEQ ID NO:278), CTLA-4BP 5-4 12c,or hB7-1-Tyr65His polypeptide variant were incubated with 2.5microgram/milliliter (ug/ml) CTLA-4-Ig or 30 ug/ml CD28-Ig (both ofwhich are saturating concentrations), and binding was determined by FACSanalysis. Panel A shows that 293 cells expressing hB7-1, CTLA-4BP5x4-12c, or hB7-1-Tyr65His polypeptide variant bind CTLA4-Ig in asubstantially similar manner. Untransfected or pCDNA3.1 transfected 293cells showed no binding to CDLA-4-Ig. However, as shown in Panel B, thebinding of each of hB7-1-Tyr65His variant and CTLA-4BP 5x4-12cpolypeptide to CD28-Ig was dramatically reduced to a level approximatingthat of untransfected or pCDNA3.1 transfected 293 cells. Cellsexpressing hB7-1 showed strong binding to CD28-Ig.

In addition to evaluating ligand binding, 293 HEK cells transfected withpCDNA or with a nucleic acid sequence encoding hB7-1 (SEQ ID NO:278),hB7-1-Tyr65His polypeptide variant, CTLA-4BP 5x4-12c (SEQ ID NO:39)(designated as clone 12c), or CD28BP-15 (SEQ ID NO:19), and 293 cellsalone (with no DNA) were assayed for their ability to induce humanT-cell proliferation (see FIG. 24). A null response was observed withthe negative controls (293 cells transfected with pCDNA and 293 cellswithout any DNA) and a robust proliferative response was observed withCD28BP-15- about 3-fold higher than cells expressing hB7-1. Cellsexpressing the hB7-1-Tyr65His variant and CTLA4-BP 5x4-12c showedproliferation responses virtually identical to those of the negativecontrol groups, indicating that T cells received little or no positiveco-stimulatory signal. The lack of T cell induced proliferation by thehB7-1-Tyr65His variant and CTLA-4BP 5x4-12c transfectants was not due toa lack of cell surface expression of these molecules, since binding ofthese molecules to CTLA4-Ig was normal (see inserted histogram in FIG.24). The data suggest the Tyr65His substitution in hB7-1 is asubstitution (mutation) that results in a polypeptide variant thatpreferentially binds to CTLA-4 relative to CD28 and/or induces adecreased level of T cell proliferation or does not induce T cellproliferation compared to the level of T cell proliferation induced byhB7-1, as determined by T cell proliferation analyses, under theconditions set forth in Example IX (FIGS. 23–24). The polypeptidevariant did not bind CD28-Ig (FIGS. 23A–23B) as did hB7-1, but it didbind CTLA-4-Ig in a manner that is substantially identical or equivalentto that of hB7-1; the binding profile of the polypeptide variant forCTLA-4-Ig was substantially identical or equivalent to that of hB7-1.The polypeptide variant has a CTLA-4/CD28 binding affinity ratio that isabout equal to or greater than the CTLA-4/CD28 binding affinity ratio ofa hB7-1, under the conditions described in FIGS. 23–24. Substitution ofan amino acid that would constitute a conservative substitution of Hisfor Tyr at this position of hB7-1 or substitution of an amino aciddifferent than His, but having functional or chemical properties similarto His (Arg, Lys, Pro, Phe, and/or Trp), may produce variants withsimilar properties.

Example X Molecules with Immunosuppressive Properties

CTLA-4-Ig has been used as an antagonist of CD28 signaling, because themolecule binds to B7-1 and B7-2 with high affinity and thereby preventsthe interaction of B7-1 and/or B7-2 with CD28 expressed on T cells.CD24-Ig also has been used in clinical trials to treat psoriasis and hasbeen shown to provide clinical benefits. To compare CTLA-4-Ig withCD28BP-15-ECD-Ig and CD28BP-15-ECD with regard to immunoregulation, weexamined the effects of these molecules on an antigen specific memory Tcell response. The ECD of CD28BP-15 (clone 15) comprises the amino acidsequence of at least about amino acids 35–244 of SEQ ID NO:66.

Blood samples from three donors were obtained from Stanford blood bank(Stanford, Calif.) (FIGS. 25A–25C). PBMCs were isolated from each ofthese donors' blood by centrifugation over Histopaque-1077 (Sigma).PBMCs were used at 1×10⁵ cells/well in 96-well round bottom assay plateand 1 ug/ml of Tetanus Toxoid (TT, Calbiochem, San Diego, Calif.) wasadded into the cultures. Increasing concentrations (ug/ml) of solubleproteins of CD28BP-15-ECD-Ig (open triangle), CD28BP-15-ECD (closetriangle), WT huB7.1-ECD-Ig (close square), WT huB7.1-ECD (open square),commercial CTLA-4Ig (R&D Systems) (closed diamond), and control humanIgG antibody (open circle) were added to the cultures as describedabove. A non-specific human IgG was used as a negative control in thisTetanus Toxoid specific response. Assay plates were incubated for totalof 5 days in 200 ul of Yssel's medium. 1uCi/well of ³H-thymidine wasadded during the last 8 hours of incubation prior to being harvested.³H-thymidine incorporation is indicative of T cell proliferation asdescribed previously. The data represent a mean of C.P.M.; each datapoint was calculated from triplicate wells. The immunosuppressiveeffects of CD28BP-15-ECD-Ig and CD28BP-15-ECD were comparable to or onlyslightly less than those of CTLA-4-Ig.

Because CTLA-4-Ig has previously been shown to provide clinical benefitin patients with psoriasis, and because the immunosuppressive effects ofCD28BP-15-ECD-Ig and CD28BP-15-ECD were comparable to or only slightlyless than those of CTLA-4-Ig, these data support the conclusion thatCD28BP-15-ECD-Ig and CD28BP-15-ECD are useful as immunosuppressiveagents. Importantly, because the inhibitory effects of CD28BP-15-ECD-Igand CD28BP-15-ECD are likely to be mediated through binding to CD28,thereby preventing (blocking) B7-1 and/or B7-2 from interacting withCD28, the interaction of B7-1 and/or B7-2 with CTLA-4 in vivo during thetreatment with CD28BP-15-ECD-Ig or CD28BP-15-ECD is likely to remainmostly unaffected. While CD28BP-15-ECD-Ig or CD28BP-15-ECD prevent thesignaling through CD28, they have little or no effect on signalingthrough CTLA-4. This is in contrast to CTLA-4-Ig, which binds to B7-1and/or B7-2, preventing the interactions of endogenous B7-1/B7-2 withboth CD28 and CTLA-4 in vivo. CD28BP-15-ECD-Ig and CD28BP-15-ECD werefound to exhibit immunosuppressive effects and are likely to be usefulin a variety of methods in which immunosuppression is desirable,including, e.g., the treatment of autoimmune diseases andtransplantation.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. For example, all thetechniques and apparatus described above may be used in variouscombinations. All publications, patents, patent applications, and/orother documents cited in this application are incorporated herein byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

SEQUENCES Clone ID SEQ ID Name Sequence SEQ ID Round 1ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:1 (R1)AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGAC CD28BP-71CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTG (CloneACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAA 71)AAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCACAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAACTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTCACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 1ATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:2CD28BP-84 TGCTCCTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCACGTCACCAAGTCGGT(Clone GAAAGAAATGGCAGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATG 84)CGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAAGTGTGGCCTGAGTACAAGAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTCCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 1ATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:3CD28BP- TGCTCTTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCAGGTCACCAAGTCGGT118 GAAAGAAATGGCAGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATG (CloneCGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAAG 118)TGTGGCCTGAGTACAAAAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 1ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:4CD28BP- AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGAC126 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTG (CloneACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAA 126)AAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:5(R2) AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCD28BP-1 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCCGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:6CD28BP-2 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCCCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACCGCCCGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:7CD28BP-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:8CD28BP-4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTGCTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:9CD28BP-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGGCATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:10CD28BP-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGAGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:11CD28BP-7 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGATGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:12CD28BP-8 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTCCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:13CD28BP-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGCGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:14CD28BP-10 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAGGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:15CD28BP-11 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACGCATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:16CD28BP-12 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:17CD28BP-13 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACTAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:18CD28BP-14 TGCTCTTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCAGGTCACCAAGTCGGTGAAAGAAATGGCGGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATGCGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAAGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:19CD28BP-15 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCCGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCACCCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:20CD28BP-16 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCACTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCGCTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:21CD28BP-17 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAAAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCCGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGTGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 1ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:22 (R1)AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA CTLA4BP-5AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 1ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:23CTLA4BP-7 AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGGGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 1ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:24CTLA4BP- AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACATGACCAAGGA11 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGTTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACACCCTGTATGA SEQ ID Round 1ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:25CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA13 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCGGGTTGGAAAATGGGGAAGAAATAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACACCCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAG SEQ ID Round 1ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:26CTLA4BP- AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA27 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGAAGGAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCACCCAAGTGTCCATACCTCAATTTCTTTC NO:27CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACTAAGGA5x2-10c AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCCGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGAC SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:28CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-11d AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCGATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGAAA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:29CTLA4BP- AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5X2-12F AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACCATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAAAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCCACTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGAAGGAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:30CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-2g AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCAACTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACCGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAC SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:31CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAGGGA5x2-3c AGTGAAAGAAGTGGCAACACTGTCCTGTGGCCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGGGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATAAG SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:32CTLA4BP- AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-4c AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGGGAAGAATTAAATGGCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGGAGGAATGAGAGACTGAGAAGGGAAAGTGTACACCCTGTATGAG SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:33CTLA4BP- GGCTCTTGGTGCTGGCTACTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-7b AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGGCTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:34CTLA4BP- AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACATGACCAAGGA5x2-8c AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCAGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAGGAATTAAATGCCATCAACACAACAGTTTCCCAAGACCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACACCCTGTATGAT SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:35CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x3-10e AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACACCCTGTATGAT SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:36CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x3-11b AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTAGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCTCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTACACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCGTACCTCAATTTCTTTC NO:37CTLA4BP- AGCTCTTGGTGCTAGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x3-6f AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTAGCACAAACTCGCATCTACTGGCAAAAGGGGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGGCCTACTGCTTTGCCCCAGGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAC SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:38CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATCTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x4-11d AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGATAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTGTCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACCGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:39CTLA4BP- AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x4-12c AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATCGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGTTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACCGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:40CTLA4BP- AGCTCTTGGTGATGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x4-1f AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTAGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGTTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGAG SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:41CTLA4BP- AGCTCTTGGTGCTAGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x5-2e AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAGATGCAGAGAGAGGAGAAGGAATGAGACACTGAGAAGGGAAAAGTGTACGCCCTGTATGAC SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:42CTLA4BP- AGCTCTTGGTGCTGGCTGGTCTTCCTCATCTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x5-6e AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCCCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGTTTTGCCCCAAGATGCAGAGAGAGAAAGAGGAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAC SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:43CTLA4BP- AGCTCCTGGTGCTGGCTGGTCTTTCTCATCTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x6-9d AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGAT SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCGTACCTCAATTTCTTTC NO:44CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x8-1f AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAGCCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAGGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAATTTCTTTC NO:45CTLA4BP- AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x9-12c AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGAG SEQ ID BaboonATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:46 B7-1AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGTTTTGCCCCAAGATGCAGAGAGAGAAGAAGGAATGAGACATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID OrangutanATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:47 B7-1AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATATGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCGGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATGATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 1MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:48CD28BP-71 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQK(Clone PVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGE 71)ELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 1MGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:49CD28BP-84 RIYWQKDQQMVLSIISGQVEVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNEN(Clone GSFRREHLTSVTLSIRADSPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNA 84)VNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round 1MGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:50CD28BP- RIYWQKDQQMVLSIISGQVEVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNEN118 GSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round 1MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:51CD28BP- TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQK126 PDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:52CD28BP-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:53CD28BP-2 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYRPACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:54CD28BP-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:55CD28BP-4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLCWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:56CD28BP-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:57CD28BP-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:58CD28BP-7 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWRSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:59CD28BP-8 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:60CD28BP-9 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWRSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:61CD28BP-10 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNASTEEL NO:62CD28BP-11 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRPNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:63CD28BP-12 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:64CD28BP-13 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:65CD28BP-14 RIYWQKDQQNVLSIISGQVEVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:66CD28BP-15 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRPSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:67CD28BP-16 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLAAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:68CD28BP-17 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTVVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round1 MGHTRRQGISPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:69CTLA4BP-5 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 1MGYTRRQGTSPSKCPYLKFFQLLVLAGLSHLCSGVIHVTNEVKEVATLSCGHNVSGEELAQ NO:70CTLA4BP-7 TRIYWQKEKKMVLTMMYGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICLTSGGFPEPRLAWMKDGEELNAISTTVSQDPGTELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFSWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 1MSHTRRQGTSPSKCPYLKFFQLLVLASLSHFCSGVIHMTKEVKEVATLSCGHNVSVEELAQ NO:71CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE11 KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTHCFAPRCRERRRNERLRRESVHPV SEQ ID Round 1MGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:72CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE13 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLFGLENGEEINAINTTASQDPETELYTVSSKLDFNMTPNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERKSNERLRRESVRPV SEQ ID Round 1MSHTRRQGISPSKCPYLNFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGLNTSVEELAQ NO:73CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE27 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNETLRRESVRPV SEQ ID Round 2MGHTRRQGISPPKCPYLNFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:74CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-10c KDAFKREHLAEVMLSVKADFPTFSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRNETLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:75CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-11d KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTDRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERKSNETLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:76CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5X2-12F KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIKRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNPLPSWAITLISANGIFVICCLTYCFAPRCRERRRNETLRRESVRPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:77CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-2g KDAFKREHLAEVMLSVKADFPTPSITDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYRFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MGYTRRQGTSPSKCPYLKFFQLLVLACLSHFCSGVIHVTREVKEVATLSCGHNVSVEELAQ NO:78CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-3c KDAFKREHLAEVMLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETGLYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MSHTRRQGTSPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:79CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE5x2-4c KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNGINTTVSQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVHPV SEQ ID Round 2MSHTRRQGISPSKCPYLNFFRLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:80CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-7b KDAFKREHLAEVTLSVKAGFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERRRNERLRRESVRFV SEQ ID Round 2MSHTRRQGTSPSKCPYLKFFQLLVLASLSHFCSGVIHMTKEVKEVATLSCGHNVSVEELAQ NO:81CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-8c KDAFKQEHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERRRNERLRRESVHPV SEQ ID Round 2MGYTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:82CTLA4BP- TRIYWQKEKKMVLTEMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECWLEYE5x3-10e KDAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERKSNERLRRESVHPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:83CTLA4BP- TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRSSDEGTYECVVLKYE5x3-11b KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAISTTVSQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRSNERLRRESVRPV SEQ ID Round 2MGHTRRQGISPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:84CTLA4BP- TRIYWQKGKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE5x3-6f KDAFKREHLAEVMLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTASQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLAYCFAPGCRERKSNERLRRESVRPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLACLSHLCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:85CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYD5x4-11d KDAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYRFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:86CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x4-12c KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVRPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVMACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:87CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x4-1F KDAFKREHLAEVMLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:88CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x5-2e KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNETLRRESVRPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLAGLPHLCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:89CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x5-6e KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAISTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISANGIFVICCLTHCFAPRCRERKRNERLRRESVRPV SEQ ID Round 2MSHTRRQGTSPSKCPYLKFFQLLVLAGLSHLCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:90CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x6-9d KDAFKREHLAEVMLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYRFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MGHTRRQGISPSKCPYLNFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:91CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x8-1f KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSASGGFPEPHLFWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIRYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MGHTRRQGTSPSKCPYLNFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:92CTLA4BP- TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x9-12c KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTASQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERKSNERLRRESVRPV SEQ ID BaboonMGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:93 B7-1TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNETLRRESVRPV SEQ ID OrangutanMGHTRRQGTSPSKCPYLNFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:94 B7-1TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRMICSTSGGFPEPHLSWLENGEELNAISTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRSNERLRRESVRPV SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:95CD28A12-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCGAAAGGATAGTAAAATGNTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGTACCATCACTGACATGAACGATAACCTCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTTGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGTGCCCATGCCTCTGGCTCTCTC NO:96CD28A4-5* AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGCTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACCGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:97CD28A4-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAAAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCCGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCTCGGGCTGAGGTACCAAGCTTAAGTTNA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:98CD28A6-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTTGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCCCTC NO:99CD28A6-1 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCACGCTTGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGATAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:100CD28A8-4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTTGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCCC NO:101CD28A8-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCCATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACAACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAGCTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:102CD28B2-8 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACGAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:103CD28B4-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:104CD28B6-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGAAAATGCAAAGTTGCTCTCAGTCTCCATGAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:105CD28B6-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGCGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTGTCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:106CD28B8-5* AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCACAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:107CD28C11-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGCGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:108CD28C6-1 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:109CD28C7-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:110CD28C8-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGGTTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:111CD28C9-5* AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCTGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTNGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:112CD28C2-4 TGCTCTTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCAGGTCACCAAGTCGGTGAAAGAAATGGCAGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATGCGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACCGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:113CD28D2-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCAGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATCCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACTGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTCGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:114CD28D2-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:115CD28D8-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:116CD28D11-1 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTGTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCTAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:117CD28D12-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAACCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGGAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:118CD28E10-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTAGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:119CD28F7-2 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCCGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATCCCTCTGGCTCTCTC NO:120CD28F8-4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCAGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:121CD28F10-2 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCCAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:122CD28F12- AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGAC5* CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGATTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGGGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGCGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:123CD28G2-8 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCCGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:124CD28G1-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAAAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCTCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATTGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:125CD28G1-9 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:126CD28H4-3 AGCTCTTGGTGCTCACTGATCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCAAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:127CD28H11-3 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGCAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:128CD28H6-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTAAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:129CD28E2-4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:130CD28B4-5a AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTCGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:131CD28A2-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGGGAAATGCAAAGTGCTCTCAGTCTCCATAGGTACCAAGCTTAAGTTTAACCGC SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:132CD28B4-5* AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTTCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:133CD28D5-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGTCCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:134CD28D10-4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:135CD28E2-5* AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCACCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCCGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:136CD28E5-2 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAGATCCTGAAACCAAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCGCAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:137CD28E8-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACACGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:138CD28E9-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGTGTTATTCAGAACCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGGCTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGGCATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:139CD28F3-1 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAAAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:140CD28F3-5 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCCGGCTCTCTC NO:141CD28F3-6 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Round2 ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:142CD28F11-8 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAACAGTGTGACCAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGTTTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGACCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:143CTLA4BP AGTTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACTAAGGA5x9-d10 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGGGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGTTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGCACGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:144CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x6-f6 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCCCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACCGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGGCTGAGAAGGGAAAGTGTATGCCCTGTATGAG SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:145CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCCCACTTCTGTTCAGGTGTTATCCACGTGACCAAGAA5c5-h12 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAATGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCGATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGGGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACAGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:146CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x5-c10 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAGCACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCCCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTCAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGAAGGAATGAGAGATTGAGAAGGGAAAGTGTACACCCTGTATGAG SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:147CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCATCTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x3-e8 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCGACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAGGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATAG SEQ ID Round 2ATGAGCCACATACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:148CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x3-c4 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCCGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGTTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:149CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACATGACCAAGGA5x3-c3 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCCCAATGTTTCCGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGTTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATAG SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCATCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:150CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGAA5x2-h11 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGGGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTGCAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGCTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGTTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAACTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCGAGTGTCCATACCTCAAGTTCTTTC NO:151CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACATGACCAAGGA5x2-d7 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCCTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGGGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:152CTLA4BP GGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-b7 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:153CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCPAGGA5x2-b1 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGCACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGGTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAGCTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGACCCTGAGAAGGGAAAGTGTACGCCCTGTATGGGGTACCAAGCTTAAGTTTAAACCGCNNATCAGCC SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:154CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACTAAGGA5x1-f1 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGCACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTCAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAACGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAATCACAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGAAGGAATGAGACACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:155CTLA4BP AGCTCTTGGTGCTAGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x1-d7 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTCCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGGGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACCTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGTTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACTTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACACCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:156CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x4-g9 AGTGAAAGAGGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGATAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCAGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCAGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTGTACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAGGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAAGGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGAGGCAGAGAGAGAAAGAGCAATGGGAGACTGAGAAGGGAAAGTGTACACCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:157CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACTAAGGA2x4-a6 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGNTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCGGTAAATGGAATTTTTGTGATATGCTGCCCGACCTACTGCTTTGCCCCAAGGTGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:158CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCTACGTGACCAAGGA2x2-f3 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGATTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATAGGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:159CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x2-f12 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGGGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGCGAATCAGACCTTCAACTCGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:160CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x1-g8 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCGTTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:161CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x1-f10 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTTAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTCTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:162CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x1-c9 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGCTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCAACTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAATGCCATCAGCACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACTAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGTTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:163CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x1-h12 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCTCAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:164CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x1-e2 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGATGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTAGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCGAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTATACACCCTGTATGA SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:165CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCATCTCTGTTCAGGTGTTATCCACGTGACTAAGGA2x1-c4 AGTGAAAGAAGTGGCAACGCTGCCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTCGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAACTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGGATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:166CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCATCTCTGTTCAGGTGTTATCCACATGACTAAGGA2x1-b12 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGCTCTGAAGTATGAAAAAGATGCTTTCAAGCAGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAGCCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTATGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:167CTLA4BP AGCTCTTGGGGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA2x2-f1 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCTATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAGGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTTCTGGCTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTACTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTCGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:168CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x4-h1 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGAAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTTCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTAATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGGAGAAGGAATGAGACACTGAGAAGGGAAAGTGTACACCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:169CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x4-a1 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGCACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGGGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:170CTLA4BP AGCTCTTGGTGCTGGCTAGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGA5x2-f3 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGCCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAGGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCCGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGGGAAGAATTAAATGCCATCAACACAACAGCTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGCAAATGGAATTTTTGTGATATGCTGCCTGACCCACTGCTTCGCCCCAAGATGCAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATAG SEQ ID Round 2ATGAGCCACACACGGAGGCAGGGAATATCACCATCCAAGTGTCCGTACCTCAAGTTCTTTC NO:171CTLA4BP AGCTCTTGGTGCTGGCTGGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACTAAGGA5x2-e12 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCCACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGGCATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTAGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCGCAGTTTTGTGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATAG SEQ ID Round 2ATGGGCTACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:172CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACTAAGGA2x4-h11 AGTGAAAGAAGTGGCAACACTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGGAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGGCCTACTGCTTTGCCCCAAGATGCAGAGGGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID Round 2ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCGTACCTCAATTTCTTTC NO:173CTLA4BP AGCTCTTGGTGCTGGCTTGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACTAAGGA2x3-h2 AGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGAGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAACCCCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGGGAAGAACTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCCTAATCTCAGTAAAGGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGGAGAGAGAGAAAGAGCAATGAGAGACTGAGAAGGGAAAGTGTACGCCCTGTATAG SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:174CD28A12-5 TSLRIYWRKDSKMXLAILPGKVQVWPEYKNRTITDMNDNLRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELLVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKCPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:175CD28A4-5* TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEKL NO:176CD28A4-9 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCPACRHVARWKRTRRNEETVGTERLSPIYLGSAQSRAEV PSLSX SEQID Round 2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEELNO:177 Cd28A6-9TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFLVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKCPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:178CD28A6-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFRVIIPVSGALVLTAIVLYCLACRHVARWKRTRNNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:179CD28A8-4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMPSCDYSTSTEEL NO:180CD28A8-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNRSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:181CD28B2-8 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:182CD28B4-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:183CD28B6-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVKMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:184CD28B6-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVIQKPVLKGAYKLEHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNATNNHSTVCLTKYGELSVSQIFPWSKPKQEPPIDQLPFWVIVPVSGALVLTAVVLYCLACRHVAR SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:185CD28B8-5* TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWPSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:186CD28C11-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIACLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:187CD28C6-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHGARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:188CD28C7-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:189CD28C8-6 TSLRIYWQKDSKNVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELGFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:190CD28C9-5* TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARXKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:191CD28C2-4 RIYWQKDQQMVLSIISGQVEVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKPEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:192CD28D2-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVIQALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRENEETVGTERLSPIYLGSAQSSG SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:193CD28D2-9 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:194CD28D8-9 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:195CD28D11-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIILVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:196CD28D12-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQPSG SEQ ID Round 2MGHTMEWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:197CD28E10-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:198CD28F7-2 TSLRIYWQKDSKNVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:199CD28F8-4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:200CD2SF10-2 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:201CD28F12- TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQK5* PDLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:202CD28G2-8 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPSINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:203CD28G1-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVSSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:204CD28G1-9 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTDLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:205CD28H4-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:206CD28H11-3 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAAVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:207CD28H6-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:208CD28E2-4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:209CD28B4-5a TSLRIYWQKDSKNVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:210CD28A2-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEGKCKVLSVSIGTKLKFNR SEQ IDRound 2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEELNO:211 CD28B4-5*TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKPDLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:212CD28D5-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQSFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:213CD28D10-4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGTTPKSVTKRVKETVMLSCDYNTSTEEL NO:214CD28E2-5* TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRPSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:215CD28E5-2 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNRSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:216CD28E8-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPETKLYMISSELDFNTTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:217CD28E9-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVIQKPDLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:218CD28F3-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGKELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:219CD28F3-5 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLRLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:220CD28F3-6 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:221CD28F11-8 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MSHTRRQGTSPSKCPYLKFFQFLVLASLSHFCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:222CTLA4 TRIYWQKGKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE5x9-d10 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTASQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTHCFAPRCRERRRNERLRRESARPV SEQ ID Round 2MGYTRRQGTSPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:223CTLA4 TPIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x6-f6 KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYRFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGYTRRQGISPSKCPYLKFFQLLVLASLSHFCSGVIHVTKKVKEVATLSCGHNVSVEELAQ NO:224CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x5-h12 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTDRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNGRLRRESVRPV SEQ ID Round 2MSHTQRQGISPSKCPYLNFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:225GTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE5x5-c10 KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAISTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVHPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLAGLSHLCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:226CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE5x3-e8 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPGCRERRRNERLRRESVCPV SEQ ID Round 2MSHIRRQGISPSKCPYLNFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:227CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x3-c4 KDAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPRLAWMEDGEELNAINTTASQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLFPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGTSPSKCPYLKFFQLLVLASLSHFCSGVIHMTKEVKEVATLSCGPNVSVEELAQ NO:228CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5c3-c3 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNCIFVICCLTHCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MSHTRRQGISSSKCPYLKFFQLLVLACLSHFCSGVIHVTKKVKEVATLSCGHNVSVEELAQ NO:229CTLA4 TRIYWQKGKKMVLTMNSGDMMIWPECKNRTIFDITNNLSIVILALRPSDEGTYECAVLKYE5x2-h11 KDAFKREHLAEVTLSVKADFPTPSTSDFEIPTSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTASQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MGYTRRQGTSPSECPYLKFFQLLVLAGLSHFCSGVIHMTKEVKEVATLSCGLNVSVEELAQ NO:230CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-d7 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETGLYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLNFFRLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:231CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-b7 KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:232CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEHKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-b1 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTGSSKLDFNMTTNHSFMCLIKYGHLRVNQTFSWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERRRNETLRRESVRPVWGTKLKFKPXIS SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:233CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEHKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x1-f1 KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTASQDPETELYTVSSKLDFNMTANHSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNETLRRESVRPV SEQ ID Round 2MGYTRRQGTSPSKCPYLNFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVPVEELAQ NO:234CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYGCVVLEYE5x1-d7 KDAFKREHLAEVMLSVKADFPTPSITDLEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTASQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERRRNERLRRESVHPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:235CTLA4 TRIYWQKDKKMVLTMMSGDMNIWPEYKNQTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x4-g9 KDAFKQEHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPRLAWMEDGEELNAISTTVSQDPGTELCTVSSKLDFNMTTNHSFMCLIRYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVKGIFVICCLTYCFAPRGRERKSNGRLRRESVHPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:236CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x4-a6 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAISTTVSQDPETELYAXSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCPTYCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIYVTKEVKEVATLSCGHNVSVEELAQ NO:237CTLA4 TRIYWQKEKKMVLIMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTECVVLKYEK2x2-f3 DAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:238CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYGCVVLEYE2x2-f12 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRANQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MGYTRRQGTSPSKCPYLNFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:239CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSVVILALRPSDEGTYECVVLKYE2x1-g8 KDAFKREHLAEVTLSVKADFPTPSITDFEIPPSNIRRIIGSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MGYTRRQGISPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:240CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x1-f10 KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYAVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGISVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:241CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTECVVLKYEK2x1-c9 KDAFKREHLAEVMLSVKADFPTPSITDFEIPTSNIRRIICSTSGGFPEPRLAWMEDGEELNAISTTASQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTHCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:242CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x1-h12 KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYCFAPRCRERKSNERLRRESVCPV SEQ ID Round 2MGYTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSDEELAQ NO:243CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x1-e2 KDAFKREHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSAITLISANGIFVICCLTYCFAPRCRERRRNERLRRESIHPV SEQ ID Round 2MGYTRRQGISPSKCPYLKFFQLLVLAGLSHLCSGVIHVTKEVKEVATLPCGHNVSVEELAQ NO:244CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x1-c4 KDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPGTELYAVSSKLDFNMTTNHNFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLVLAGLSHLCSGVIHMTKEVKEVATLSCGHNVSVEELAQ NO:245CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVALKYE2x1-b12 KDAFKQEHLAEVTLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPRLAWMEDGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVCPV SEQ ID Round 2MGHTRRQGISPSKCPYLKFFQLLGLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:246CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRLSDEGTYECVVLKYE2x2-f1 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTASQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:247CTLA4 TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRLSDEGTYECVVLKYE5x4-h1 KDAFKRKHLAEVMLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTASQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPNNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNETLRRESVHPV SEQ ID Round 2MGHTRRQGTSPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:248CTLA4 TRIYWQKEKKMVLTMNSGDMNIWPEHKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x4-a1 KDAFKREHLAEVTLSVKADFPTPSITDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLASLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:249CTLA4 TRIYWQKEKKMVLTMMPGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLRYE5x2-f3 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTASQDPETELYTVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTHCFAPRCRERKSNERLRRESVRPV SEQ ID Round 2MSHTRRQGISPSKCPYLKFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:250CTLA4 TRIHWQKEKKMVLTMMSGGMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE5x2-e12 KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAISTTVSQDPGTELYAVSSKLDFNMTTNRSFVCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID Round 2MGYTRRQGTSPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:251CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLEYE2x4-h11 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPGTELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLAYCFAPRCRGRRRNERLRRESVRPV SEQ ID Round 2MGHTRRQGTSPSKCPYLNFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:252CTLA4 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE2x3-h2 KDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTPGGFPEPRLAWMEDGEELNAISTTVSQDPGTELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVKGIFVICCLTYCFAPRWRERKSNERLRRESVRPV SEQ ID Round 2ATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:253CTLA4 TGCTCTTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCAGGTCACCAAGTCGGTA-H3-6 GAAAGAAATGGCAGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATGCGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAAGTGTGGCCTGAGTACAAAAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCCTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGAG SEQ ID Round 2ATGGGTCACACAATGGAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:254CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGCGACA-B11-5 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATAG SEQ ID Round 2ATGGGCCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:255CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-E2-6 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGCCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:256CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-F1-6 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAGATGCAAAGTTGCTCTCAGTCTCCATAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:257CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-F6-9 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:258CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-H4-5* CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACAGTTTCCCAAGATCCTGGAACTGAGCTCTACATGATTAGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round 2ATGGATCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:259CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-B4-6 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAACGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGCTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTTCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCGGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATAG SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:260CTLA4 AGCTCTTGGTGCCCACTGGTCTTTTTTACTTCTGTTCAGGTATCACCCCAAAGAGTGTGACA-F10-1 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTCGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCCTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAGTGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:261CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-G8-1 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGTATTGTGATCCTGGCTCTGCCCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCAGAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAACTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCACTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAAGGGAGAATGAAGTGGAAATGCAAAGTTGCTCTCAGTCTCCATGA SEQ ID Round 2ATGGGTCACACAATGAAGTGGGGATCACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:262CTLA4 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGACA-C9-9 CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAGCACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAGTACAAAAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAGGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGCCCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCGGCAGTGAACTGGATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCCGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGAG SEQ ID Round 2MGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:263CTLA4 RIYWQKDQQMVLSIISGQVEVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNENA-H3-6 GSFRREHLTSVTLSIPADFPVPSINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMEWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSATKRVKETVMLSCDYSTSTEEL NO:264CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKA-B11-5 PDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:265CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVVQKA-E2-6 NENGSFRREHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLPQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVENQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:266CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKA-F1-6 NENGSFRREHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:267CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKA-F6-9 PDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:268CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKA-H4-5* NENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLYWLENGEELNATNTTVSQDPGTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Round2 MDHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:269CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKA-B4-6 NENGSFRREHLTSVTLSIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVPTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:270CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDKGTYTCVVQKA-F10-1 NENGSFRREHLTSVTLSIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTLYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVENQSCSQSP SEQ ID Round 2MGHTVKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:271CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALPLSDSGTYTCVIQKA-G8-1 PDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID Round 2MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYSTSTEEL NO:272CTLA4 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTFPDIINNLSLMILALRLSDRGTYTCVVQKA-C9-9 NENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVGSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLARRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID HumanB7- ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTC NO:2731 AGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCTGAGCCTCACCTCTCCTGGCTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID RhesusATGGGCCACACACGCAGGGAGGAAATATCACCATCCAAGTGTCCATACCTCAAGTTCTTTC NO:274B7-1 AGCTCTTGGTGCTGGCTTGTCTTTCTCATTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGCCCCGAGTACAAGAACCGGACCATCTTTGATATCACAAATAACCTCTCCATTGTGATTCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGATGCTTTCAAGCGGGAACACCTGGCTGAAGTGATGTTATCCGTCAAAGCTGACTTCCCTACACCTAGTATAACTGACTCTGAAATTCCACCTTCTAACATTAGAAGGATAATTTGCTCAAACTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAGCACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATACTGTTAGCAGCAAACTGGATTTCAATATGACAACCAATCACAGTTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAACACACCCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTATCCTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGTTTTGCCCCAAGGTGCAGAGAGAGAAGAAGGAATGAGACATTGAGAAGGGAAAGTGTACGCCCTGTATGA SEQ ID BovineATGGGTCACACAATGAAGTGGGGAACACTACCACCCAAGCGCCCATGCCTCTGGCTCTCTC NO:275B7-1 AGCTCTTGGTGCTCACTGGTCTTTTTTACTTCTGTTCAGGCATCACCCCAAAGAGTGTGAC (cow)CAAAAGAGTGAAAGAAACAGTAATGCTATCCTGTGATTACAACACATCCACTGAAGAACTGACAAGCCTTCGGATCTATTGGCAAAAGGATAGTAAAATGGTGCTGGCCATCCTGCCTGGAAAAGTGCAGGTGTGGCCTGAATACAAGAACCGCACCATCACTGACATGAACGATAACCCCCGCATTGTGATCCTGGCTCTGCGCCTGTCGGACAGTGGCACCTACACCTGTGTTATTCAGAAGCCTGATTTGAAAGGGGCTTATAAACTGGAGCACCTGACTTCCGTGAGGTTAATGATCACAGCTGACTTCCCTGTCCCTACCATAAATGATCTTGGAAATCCATCTCCTAATATCAGAAGGCTAATTTGCTCAACCTCTGGAGGTTTTCCAAGGCCCCACCTCTACTGGTTGGAAAATGGAGAAGAATTAAATGCTACCAACACAACACTGTCCCAAGATCCTGAAACCAAGCTCTACATGATTAGCAGTGAACTGGATTTCAACATGACAAGCAATCACAGCTTCTTGTGTCTTGTCAAGTATGGAGACTTAACAGTGTCACAGACCTTCTACTGGCAAGAATCCAAACCAACCCCTTCTGCTAATCAGCACCTGACCTGGACCATTATTATCCCAGTCTCAGCATTTGGGATTTCTGTGATCATTGCAGTTATACTAACATGCCTGACCTGCAGAAATGCTGCAATACGCAGACAGAGAACGGAGAATGAAGTGGAAATGGAAAGTTGCTCTCAGTCTCCA SEQ ID RabbitATGGGCCACACGCTGAGGCCGGGAACTCCACTGCCCAGGTGTCTACACCTCAAGCTCTGCC NO:276B7-1 TGCTCTTGGCGCTGGCGGGTCTCCACTTCTCTTCAGGTATCAGCCAGGTCACCAAGTCGGTGAAAGAAATGGCAGCACTGTCCTGTGATTACAACATTTCTATCGATGAACTGGCGAGAATGCGCATATACTGGCAGAAGGACCAACAGATGGTGCTGAGCATCATCTCTGGGCAAGTGGAAGTGTGGCCTGAGTACAAGAACCGCACCTTCCCCGACATCATTAACAACCTCTCCCTTATGATCCTGGCACTGCGCCTGTCGGACAAGGGCACCTACACCTGCGTGGTTCAGAAGAATGAGAACGGGTCTTTCAGACGGGAGCACCTGACCTCCGTGACACTGTCCATCAGAGCTGACTTCCCTGTCCCTAGCATAACTGACATTGGACATCCCGACCCTAATGTGAAAAGGATAAGATGCTCCGCCTCTGGAGGTTTTCCAGAGCCTCGCCTCGCCTGGATGGAAGATGGAGAAGAACTAAACGCCGTCAACACGACGGTTGACCAGGATTTGGACACGGAGCTCTACAGCGTCAGCAGTGAACTGCATTTCAATGTGACAAATAACCACAGCATCGTGTGTCTCATCAAATACGGGGAGCTGTCGGTGTCACAGATCTTCCCTTGGAGCAAACCCAAGCAGGAGCCTCCCATTGATCAGCTTCCATTCTGGGTCATTATCCCAGTAAGTGGTGCTTTGGTGCTCACTGCGGTAGTTCTCTACTGCCTGGCCTGCAGACATGTTGCGAGGTGGAAAAGAACAAGAAGGAATGAAGAGACAGTGGGAACTGAAAGGCTGTCCCCTATCTACTTAGGCTCTGCGCAATCCTCGGGCTGA SEQ ID Cat B7-1ATGGGTCACGCAGCAAAGTCGAAAACACCACTACTGAAGCACCCATATCCCAAGCTCTTTC NO:277CGCTCTTGATGCTAGCTAGTCTTTTTTACTTCTGTTCAGGTATCATCCAGGTGAACAAGACAGTGGAAGAAGTAGCAGTACTATCCTGTGATTACAACATTTCCACCAAAGAACTGACGGAAATTCGAATCTATTGGCAAAAGGATGATGAAATGGTGTTGGCTGTCATGTCTGGCAAAGTACAAGTGTGGCCCAAGTACAAGAACCGCACATTCACTGACGTCACCGATAACCACTCCATTGTGATCATGGCTCTGCGCCTGTCAGACAATGGCAAATACACTTGTATTATTCAAAAGATTGAAAAAGGGTCTTACAAAGTGAAACACCTGACTTCGGTGATGTTATTGGTCAGAGCTGACTTCCCTGTCCCTAGTATAACTGATCTTGGAAATCCATCTCATAACATCAAAAGGATAATGTGCTTAACTTCTGGAGGTTTTCCAAAGCCTCACCTCTCCTGGCTGGAAAATGAAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTACACTATTAGCAGTGAACTGGATTTCAATATGACAAACAACCATAGCTTCCTGTGTCTTGTCAAGTATGGAAACTTACTAGTATCACAGATCTTCAACTGGCAAAAATCAGAGCCACAGCCTTCTAATAATCAGCTCTGGATCATTATCCTGAGCTCAGTAGTAAGTGGGATTGTTGTGATCACTGCACTTACCTTAAGATGCCTAGTCCACAGACCTGCTGCAAGGTGGAGACAAAGAGAAATGGGGAGAGCGCGGAAATGGAAAAGATCTCACCTGTCTACATAGATTCTGCAGAACCACTGTATGCAGAGCATCTGGAGGTAGCCTCTTTAGCTCTTCTCTACTAG SEQ ID Human B7-MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:278 1(signal TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEsequence KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNunder- AINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLlined) PSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID RhesusMGHTRRQGISPSKCPYLKFFQLLVLACLSHLCSGVIHVTKEVKEVATLSCGHNVSVEELAQ NO:279B7-1 TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPRLSWLENGEELNAISTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTHCFAPRCRERRRNETLRRESVRPV SEQ ID BovineMGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:280B7-1 TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID RabbitMGHTLRPGTPLPRCLHLKLCLLLALAGLHFSSGISQVTKSVKEMAALSCDYNISIDELARM NO:281B7-1 RIYWQKDQQMVLSIISGQVEVWPEYKNRTFPDIINNLSLMILALRLSDKGTYTCVVQKNENGSFRREHLTSVTLSIRADFPVPSITDIGHPAPNVKRIRCSASGGFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ ID Cat B7-1MGHAAKWKTPLLKHPYPKLFPLLMLASLFYFCSGIIQVNKTVEEVAVLSCDYNISTKELTE NO:282IRIYWQKDDEMVLAVMSGKVQVWPKYKNRTFTDVTDNHSIVIMALRLSDNGKYTCIIQKIEKGSYKVKHLTSVMLLVRADFPVPSITDLGNPSHNIKRTMCLTSGGFPKPHLSWLENEEELNAINTTVSQDPETELYTISSELDFNMTNNHSFLCLVKYGNLLVSQIFNWQKSEPQPSNNQLWIIILSSVVSGIVVITALTLRCLVHRPAARWRQREMGRARKWKRSHLST SEQ ID CD28BPMGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:283Consensus TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGEELNATNTTVSQDPDTELYMISSELDFNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLTAVVLYCLACRHVARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ IDCD28BP MGHTMXWXSLPPKXPCLXXXQLLVLTXLFYFCSGITPKSVTKRVKETVMLSCDYXTSTEXLNO:284 CGformCTSLRIYWXXDSKMVLAILPGKVQVWPEYKNRTITDMNDNXRIVIXALRXSDXGTYTCVXQKPXLKGAYKLEHLXSVRLMIRADFPVPXXXDLGNPSPNIRRLICSXXXGFPRPHLXWLENGEELNATNTTXSQDPXTXLYMISSELXFNVTNNXSIXCLIKYGELXVSQIFPWSKPKQEPPIDQLPFXVIIPVSGALVLXAXVLYXXACRHXARWKRTRRNEETVCTERLSPIYLGSAQSSG SEQ IDCD28BP MGHTMKWRSLPPKRPCLWPSQLLVLTDLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEELNO:285 CG1cTSLRIYWQKDSKNVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRPSDKGTYTCVVQKPVLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRFHLCWLENGEELNATNTTVSQDPGTELYMISSELGFNVTNNHSIACLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIIPVSGALVLAAVVLYRPACRHGARWKRTRRNEETVGTERLSPIYLGSAQSSG SEQ IDCTLA4BP MGHTRRQGISPSKCPYLKFFQLLVLACLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQNO:286 ConsensusTRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVMLSVKADFPTPSISDFEIPPSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV SEQ ID CTLA4BPMGHTRRQGTSPSKCPYLKFFQLLVXACLXHLCSGVIHVTXEVKEVATLSCGHNVSVEELAQ NO:287CGform TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYXKDAFKRXHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSXSGGFPEPHLFWLENGEELNAINTTVSQDPETXLYTVSSKLDFNMTANHSFMCLIXYGHLRVNQTFNWNTPKQEHFPXNLLPSWAITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV SEQ ID CTLA4BPMGHTRRQGTSPPECPYLKFFQLLVMACLPHLCSGVIHVTREVKEVATLPCGLNVSVEELAQ NO:288 CG1TPIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYDKDAFKQKHLAEVMLSVKADFPTPSITDFEIPPSNIKRIICSASGGFPEPHLFGLENGEEINAINTTVSQDPETGLYTVSSKLDFNMTADHNFMCLIRYGHLRVNQTFNWNTPKQEHFPNNPLPSWAITLISANGIFVICCPTYRFAPGCRERKSNETLRRESVCPV SEQ ID CTLA4BPMGHTRRQGTSPSKCPYLKFFQLLVLACLSHLCSGVIHVTKEVKEVATLSCGLNVSVEELAQ NO:289 CG2TRIHWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVMLSVKADFPTPSITDFEIPPSNIRRIICSTSGGFPEPHLFWLENGEELNAINTTVSQDPETELYTVSSKLDFNMTANHSFMCLIKYGHLRVNQTFNWNTPKQEHFPDNLLPSWAITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV SEQ ID CD28BPMGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMXSCDYXXSTEEL NO:290CGformD TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDXGTYTCVXQKXXXXGXXXXEHLXSVXLXIRADFPVPSITDIGHPAPNVKRIRCSASGXFPEPRLAWMEDGEELNAVNTTVXXXLDTELYSVSSELDXNXTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIXXVSGALVLTAVVLYCLACRHVAR SEQ ID CD28BPMGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNASTEEL NO:291CG1d TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPVLKGAYKLEHLASVRLMIRADFPVPSITDIGHPAPNVKRIRCSASGDFPEPRLAWMEDGEELNAVNTTVDQDLDTELYSVSSELDSNVTNNHSIVCLIKYGELSVSQIFPWSKPKQEPPIDQLPFWVIILVSGALVLTAVVLYCLACRHVAR SEQ ID CD28BPMGHTMKWGXLPPKRPCLWLSQLLVLTGLFYFCSGXTPKSVTKRVKETVMLSCDYXTSTEEL NO:292CGformB TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRXSDSGTYTCVIQKPXLKGAYKLEHLXSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGXELNATNTTXSQDPETKLYMISSELDFNXTSNXXXLCLVKYGDLTVSQXFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVXNXSCSQSP SEQ ID CD28BPMGHTMKWGTLPPKRPCLWLSQLLVLTGLFYFCSGITPKSVTKRVKETVMLSCDYNTSTEEL NO:293CG1b TSLRIYWQKDSKMVLAILPGKVQVWPEYKNRTITDMNDNPRIVILALRLSDSGTYTCVIQKPDLKGAYKLEHLTSVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENGKELNATNTTLSQDPETKLYMISSELDFNMTSNHSFLCLVKYGDLTVSQTFYWQESKPTPSANQHLTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVKMESCSQSP

1. An isolated or recombinant polypeptide variant of an extracellulardomain of a wild-type primate B7-1 comprising a polypeptide sequencethat has at least 95% identity to the polypeptide sequence of theextracellular domain of the wild-type primate B7-1 and differs from thepolypeptide sequence of the extracellular domain of the wild-typeprimate B7-1 by the substitution of an amino acid other than alanine atan amino acid residue position corresponding to position 65 of thepolypeptide sequence of wild-type human B7-1 (SEQ ID NO:278), whereinsaid polypeptide variant has a CTLA-4/CD28 binding affinity ratiogreater than the CTLA-4/CD28 binding affinity ratio of the extracellulardomain of the wild-type primate B7-1.
 2. The polypeptide variant ofclaim 1, wherein the substituted amino acid is selected from the groupconsisting of histidine, arginine, lysine, proline, phenylalanine, andtryptophan.
 3. The polypeptide variant of claim 1, wherein the primateB7-1 is human B7-1.
 4. The polypeptide variant of claim 2, wherein thesubstituted amino acid is histidine.
 5. The polypeptide variant of claim2, wherein the substituted amino acid is histidine.
 6. The polypeptidevariant of claim 3, wherein the variant has a CTLA-4/CD28 bindingaffinity ratio greater than the CTLA-4/CD28 binding affinity ratio ofthe extracellular domain of wild-type human B7-1.
 7. The polypeptidevariant of claim 1, wherein the variant induces less T cellproliferation compared to T cell proliferation induced by theextracellular domain of wild-type primate B7-
 1. 8. The polypeptidevariant of claim 5, wherein the variant has a CTLA-4/CD28 bindingaffinity ratio greater than the CTLA-4/CD28 binding affinity ratio ofthe extracellular domain of wild-type human B7-1 and/or induces less Tcell proliferation compared to T cell proliferation induced by theextracellular domain of wild-type human B7-1.
 9. The polypeptide variantof claim 1, wherein the polypeptide comprises a fusion proteincomprising at least one additional amino acid sequence.
 10. Thepolypeptide variant of claim 9, wherein the at least one additionalamino acid sequence comprises at least one Ig polypeptide.
 11. Thepolypeptide variant of claim 10, wherein the at least one Ig polypeptidecomprises at least one human IgG polypeptide comprising an Fc hinge, aCH2 domain, and a CH3 domain.
 12. The polypeptide variant of claim 8,which further comprises at least one Ig polypeptide.
 13. A multimercomprising at least two polypeptide variants of claim
 1. 14. A multimercomprising at least two polypeptide variants of claim
 5. 15. An isolatedor recombinant polypeptide variant of a mature domain of a wild-typeprimate B7-1 comprising a polypeptide sequence that has at least 95%identity to the polypeptide sequence of the mature domain of thewild-type primate B7-1 and differs from the polypeptide sequence of themature domain of the wild-type primate B7-1 by the substitution of anamino acid other than alanine at an amino acid residue positioncorresponding to position 65 of the polypeptide sequence of wild-typehuman B7-1 (SEQ ID NO:278), wherein said polypeptide variant has aCTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CD28 bindingaffinity ratio of the mature domain of the wild-type primate B7-1. 16.The polypeptide variant of claim 15, wherein the substituted amino acidis selected from the group consisting of histidine, arginine, lysine,proline, phenylalanine, and tryptophan.
 17. The polypeptide variant ofclaim 15, wherein the primate B7-1 is human B7-1.
 18. The polypeptidevariant of claim 17, wherein the substituted amino acid is histidine.19. The polypeptide variant of claim 17, wherein the variant has aCTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CD28 bindingaffinity ratio of the mature domain of wild-type human B7-1.
 20. Thepolypeptide variant of claim 15, wherein the variant induces less T cellproliferation compared to T cell proliferation induced by the maturedomain of wild-type primate B7-1.
 21. The polypeptide variant of claim18, wherein the variant has a CTLA-4/CD28 binding affinity ratio greaterthan the CTLA-4/CD28binding affinity ratio of the mature domain ofwild-type human B7-1 and/or induces less T cell proliferation comparedto T cell proliferation induced by the mature domain of wild-type humanB7-1.
 22. An isolated or recombinant polypeptide variant of a wild-typeprimate B7-1 comprising a polypeptide sequence that has at least 95%identity to the full-length polypeptide sequence of the wild-typeprimate B7-1 and differs from the polypeptide sequence of the wild-typeprimate B7-1 by the substitution of an amino acid other than alanine atan amino acid residue position corresponding to position 65 of thesequence of human B7-1 (SEQ ID NO:278), wherein said polypeptide varianthas a CTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CD28binding affinity ratio of the wild-type primate B7-1.
 23. Thepolypeptide variant of claim 22, wherein the substituted amino acid isselected from the group consisting of histidine, arginine, lysine,proline, phenylalanine, and tryprophan.
 24. The polypeptide variant ofclaim 23, wherein the primate B7-1 is human B7-1.
 25. The polypeptidevariant of claim 24, wherein the substituted amino acid is histidine.26. The polypeptide variant of claim 25, wherein the variant has aCTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CD28 bindingaffinity ratio of wild-type human B7-1.
 27. The polypeptide variant ofclaim 25, wherein the variant induces less T cell proliferation comparedto T cell proliferation induced by the wild-type primate B7-1.
 28. Acomposition comprising the polypeptide variant of claim 1 and apharmaceutically acceptable excipient or carrier.
 29. A compositioncomprising the polypeptide variant of claim 8 and a pharmaceuticallyacceptable excipient or carrier.
 30. A composition comprising thepolypeptide variant of claim 15 and a pharmaceutically acceptableexcipient or carrier.